Inhibitors of MAdCAM-1-mediated interactions and methods of use therefor

ABSTRACT

The present invention provides novel compounds comprising peptide sequences which mimic the conserved amino acid motif LDTSL of MAdCAM-1 and which have groups bonded to the N- and C-termini. Also provided are methods of inhibiting the interaction of a cell bearing a ligand of MAdCAM-1, such as human α4β7, with MAdCAM-1 or a portion thereof (e.g., the extracellular domain), comprising contacting the cell with a compound of the present invention.

RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. Ser. No. 09/109,879filed on Jul. 2, 1998, which is a Continuation of InternationalApplication No. PCT/US97/00291₁ filed on Jan. 3, 1997, which designatesthe United States, and which is a Continuation-In-Part of U.S. Ser. No.08/582,740, filed Jan. 4, 1996, now U.S. Pat. No. 6,037,324, theteachings of all of which are incorporated herein by reference in theirentirety.

GOVERNMENT SUPPORT

[0002] Work described herein was supported in whole or in part byGovernment Grant No. 43DK8498301. The government may have certain rightsin this invention.

BACKGROUND OF THE INVENTION

[0003] Lymphocyte homing from the circulation to the lymphoid tissuesand migration to sites of inflammation is regulated by interaction withreceptors expressed in postcapillary venules, including high endothelialvenules (HEV) found in secondary lymphoid tissues (e.g., mesentericlymph nodes, Peyer's Patches (PP)) (Bevilacqua, M. P., Annu. Rev.Immunol., 11:767-804 (1993); Butcher, E. C., Cell, 67: 1033-1036 (1991);Picker, L. J., et al., Annu. Rev. Immunol., 10:561-591 (1992); andSpringer, T. A., Cell, 76: 301-314 (1994)). These interactions aretissue specific in nature.

[0004] Inflammation (e.g., chronic inflammation) is characterized byinfiltration of the affected tissue by leukocytes, such as lymphocytes,lymphoblasts, and mononuclear phagocytes. The remarkable selectivity bywhich leukocytes preferentially migrate to various tissues during bothnormal circulation and inflammation results from a series of adhesiveand activating events involving multiple receptor-ligand interactions asproposed by Butcher and others (Butcher, E. C., Cell, 67: 1033-1036(1991); vonAdrian, U. H. , et al., Proc. Natl. Acad. Sci. USA, 88:7538(1991); Mayadas, T. N., et al., Cell, 74:541 (1993); (Springer, T. A. ,Cell, 76:301 (1994)). As an initial step, there is a transient, rollinginteraction between leukocytes and endothelium, which results from theinteraction of selectins (and by α4 integrins in some instances) withtheir carbohydrate ligands. This interaction, which is characterized byrolling in the direction of flow, can be assessed by known methods(Lawrence, M. B. and T. A. Springer, Cell, 65:859 (1991); WO 92/21746,Springer et al., (Dec. 10, 1992)). This is followed by activation eventsmediated by chemoattractants such as chemokines and their receptors,which cause activation of integrin adhesiveness and influence thedirection of migration of leukocytes through vascular walls. Suchsecondary signals in turn trigger the firm adhesion of leukocytes toendothelium via leukocyte integrins and their endothelial ligands(Ig-like receptors and the ECM), and subsequent transendothelialmigration from the circulation across the vascular endothelium.

[0005] In secondary lymphoid tissues, such as Peyer's patches (PPs) andlymph nodes (e.g., peripheral lymph nodes (PLN)), leukocyte traffickingand homing is regulated by interactions of homing receptors on thesurface of leukocytes with endothelial cells lining the post-capillaryvenules, notably high endothelial venules (HEV) (Gowans, J. L. and E. J.Knight, Proc. R. Soc. Lond., 159:257 (1964)). Receptors termed vascularaddressing, which are present on the endothelial cell surface andregulate the migration and subsequent extravasatlon of lymphocytesubsets. The vascular addressing show restricted patterns of expressionand this tissue specific expression makes an important contribution tothe specificity of leukocyte trafficking (Picker, L. J. and E. C.Butcher, Annu. Rev. Immunol., 10:561-591 (1992); Berg, E. L., et al.,Cellular and molecular mechanisms of inflammation, 2:111 (1991);Butcher, E. C., Cell, 67:1033-1036 (1991)).

[0006] Mucosal vascular addressin MAdCAM-1 (Mucosal Addressin CellAdhesion Molecule-1) is an immunoglobulin superfamily adhesion receptorfor lymphocytes, which is distinct from VCAM-1 and ICAM-1. MAdCAM-1 wasidentified in the mouse as a −60 kd glycoprotein which is selectivelyexpressed at sites of lymphocyte extravasation. In particular, MAdCAM-1expression was reported in vascular endothelial cells of mucosaltissues, including gut-associated tissues or lymph of the small andlarge intestine, and the lactating mammary gland, but not in peripherallymph nodes. MAdCAM-1 is involved in lymphocyte binding to Peyer'sPatches. (Streeter, P. R., et al., Nature, 331:41-46 (1988); Nakache,M., et al., Nature, 337: 179-181 (1989); Picker, L. J., et al., Annu.Rev. Immunol., 10:561-591 (1992); Briskin, M. J., et al., Nature,363:461 (1993); Berg, E. L., et al., Nature, 366:695-698 (1993); Berlin,C., et al., Cell, 74:185-195 (1993)). MAdCAM-1 can be induced in vitroby proinflammatory stimuli (Sikorski, E. E., et al., J. Imnunol.,151:5239-5250 (1993)). cDNA clones encoding murine and primate (e.g.,human) MAdCAM-1 have been isolated and sequenced (Briskin, M. J. et al.,Stature, 363: 461-464 (1993); Briskin et al., WO 96/24673, publishedAug. 15, 1996; and Briskin, M. J. et al., U.S. Ser. No. 08/523,004,filed Sep. 1, 1995, the teachings of each of which is incorporatedherein by reference in its entirety).

[0007] MAdCAM-1 specifically binds the lymphocyte integrin α4β7 (alsoreferred to as LPAM-1 (mouse), α4βp (mouse)), which is a lymphocytehoming receptor involved in homing to Peyer's patches (Berlin, C., etal., Cell, 80:413-422 (1994); Berlin, C., et al., Cell, 74:185-195(1993); and Erle, D. J., et al., J. Immunol., 153: 517-528 (1994)). Incontrast to VCAM-1 and fibronectin, which interact with both α4β1 andα4β7 (Berlin, C., et al., Cell, 74: 185-195 (1993); Strauch, U. S., etal., Int. Immunol., 6:263 (1994)), MAdCAM-1 is a selective ligand forα4β7 receptor.

[0008] Inflammatory bowel disease (IBD), such as ulcerative colitis andCrohn's disease, for example, can be a debilitating and progressivedisease involving inflammation of the gastrointestinal tract. Affectingan estimated two million people in the United States alone, symptomsinclude abdominal pain, cramping, diarrhea and rectal bleeding. IEDtreatments have included anti-inflammatory drugs (such ascorticosteroids and sulfasalazine), immunosuppressive drugs (such as6-mercaptopurine, cyclosporine and azathioprine) and surgery (such as,colectomy). Podolsky, New Engl. J. Med., 325:928-937 (1991) andPodolsky, New Engl. J. Med., 325:1008-1016 (1991). There is a need forinhibitors of MAdCAM-1 function to provide new therapies useful in thetreatment of IED and other diseases involving leukocyte infiltration ofthe gastrointestinal tract or other mucosal tissues.

SUMMARY OF THE INVENTION

[0009] As shown herein, the conserved amino acid motif LDTSL (SEQ IDNO:1) is involved in Mucosal Addressin Cell Adhesion Molecule-1(hereinafter “MAdCAM-1”) binding to MAdCAM-1 ligands, such as humanα4β7. In addition, compounds containing this peptide sequence ortruncated versions thereof, e.g., Asp-Thr and Leu-Asp-Thr, bind to α4β7and can inhibit adhesion of leukocytes expressing α4β7 on the cellsurface to MAdCAM-1. It has also been discovered that groups on the N-and C-termini of the peptide sequences enhance the binding of thesecompounds to α4β7 and are more potent inhibitors of interaction betweenMAdCAM-1 and its ligands. Accordingly, the present invention providesnovel compounds comprising peptide sequences which mimic the conservedamino acid motif LDTSL and which have groups bonded to the N- andC-termini.

[0010] Also provided are methods of inhibiting the interaction of a cellbearing a ligand of MAdCAM-1, including α4β7 integrins, with MAdCAM-1 ora portion thereof (e.g., the extracellular domain), comprisingcontacting the cell with a compound of the present invention. In oneembodiment, the invention relates to a method of inhibiting theMAdCAM-mediated interaction of a first cell bearing an α4β7 integrinwith MAdCAM, for example with a second cell bearing MAdCAM, comprisingcontacting the first cell with a compound of the present inventLon. Inanother embodiment, the invention relates to a method of treating anindividual suffering from a disease associated with leukocyterecruitment to tissues (e.g., endothelium) expressing the moleculeMAdCAM-1.

[0011] One embodiment of the present invention is a method of inhibitingthe binding of a cell such as a leukocyte expressing a ligand forMAdCAM-1 on the cell surface (e.g., α4β7) to MAdCAM-1, for example toendothelial cells expressing MAdCAM-1 on the cell surface. The methodcomprises contacting the leukocytes with an effective amount of aninhibitor represented by Structural Formula (I):

R¹—X—Y—Z—R²  (I)

[0012] Y is a pentapeptide [AA]₁-[AA]₂-[AA]₃-[AA]₄—[AA]₅.

[0013] [AA]₁ is selected from the group consisting of leucine, valine,isoleucine, alanine, phenylalanine, glycine, N-methylleucine, serine,threonine, ornithine, cysteine, aspartic acid, glutamic acid and lysine.

[0014] [AA]₂ is selected from the group consisting of aspartic acid,glutamic acid, phenylalanine and tyrosine.

[0015] [AA]₃ is selected from the group consisting of threonine, serine,valine, proline and 4-hydroxyproline.

[0016] [AA]₄ is selected from the group consisting of serine, cysteine,aspartic acid, glutamic acid, proline, 4-hydroxyproline, threonine,valine, isoleucine, alanlne, glycine, ornithine and lysine.

[0017] [AA]₅ is selected from the group consisting of leucine, valine,isoleucine, N-methylleucine, threonine, ornithine, serine, alanine,glycine, phenylalanine, cysteine, aspartic acid, glutamic acid andlysine.

[0018] X and Z are independently chosen from the group consisting of acovalent bond, an amino acid or a peptide. Each amino acid in X and Z isindependently selected from the group of naturally occurring aminoacids. X and Z are preferably covalent bonds.

[0019] R¹ is R³—CO—.

[0020] R² is —NR⁴R .

[0021] R³ is selected from the group consisting of a lower alkyl group,a substituted lower alkyl group, an aryl group, a substituted arylgroup, a heteroaryl group and a substituted heteroaryl group.

[0022] R⁴ and R⁵ are each independently selected from the groupconsisting of hydrogen, a lower alkyl group, a substituted lower alkylgroup, an aryl group, a substituted aryl group, a heteroaryl group and asubstituted heteroaryl group. R⁴ and R⁵ are not both —H. Taken together,R⁴ and R⁵ can also form a heterocyclic ring.

[0023] Taken together, X, Y and Z form a peptide containing no more thanabout fifteen amino acid residues.

[0024] The compound represented by Structural Formula (I) is a peptidehaving a group bonded to the nitrogen atom at the N-terminus and asecond group bonded to the carbonyl at the C-terminus. In one aspect,the peptide is linear. The N-terminus of Y is not bonded to arginine oran arginine derivative.

[0025] Optionally, the peptide formed by X, Y and Z is cyclized to forma ring. When cyclized, the ring can be formed by an amide linkage, anester linkage or a dlsulfide linkage between two amino acids in thepeptide. When the ring is formed by an amide linkage between theN-terminus and C-terminus of the peptide, R¹ and R² together form acovalent bond between the carbonyl at the C-terminus of the peptide andthe nitrogen at the N-terminus of the peptide.

[0026] In another embodiment of the method of inhibiting the binding ofa cell such as a leukocyte expressing a ligand for MAdCAM-1 on the cellsurface (e.g., α4β7) to MAdCAM-1, for example endothelium expressing themolecule MAdCAM-1, the inhibitor administered is represented byStructural Formula (II):

R¹—X—Y′—Z—R²  (II)

[0027] R¹, R², X and Z are as defined for Structural Formula (I). Y′represents a dipeptide having the sequence Asp-Thr, a tripeptide havingthe sequence Leu-Asp-Thr, or a pentapeptide[AA]₁-[AA]₂-[AA]₃-[AA]₄-[AA]₅ having the sequence Leu-Asp-Thr-Ser-Leu(SEQ ID NO:1) with the proviso that any single one of [AA]₁, [AA]₂,[AA]₃, [AA]₄ or [AA]₅ can vary, being any naturally occurring aminoacid. For example, the pentapeptide can be Xaa-Asp-Thr-Ser-Leu (SEQ IDNO.: 85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO: 86), Leu-Asp-Xaa-Ser-Leu (SEQID NO: 87), Leu-Asp-Thr-Xaa-Leu (SEQ ID NO: 88), or Leu-Asp-Thr-Ser-Xaa(SEQ ID NO: 89). The nitrogen at the N-terminus of Y′ may be bonded toany naturally occurring amino acid (including arginine) with the provisothat the N-terminus of Y′ may not be bonded to glycine or sarcosine whenY′ is Asp-Thr and the peptide formed from X—Y′—Z is cyclized, asdescribed below.

[0028] X, Y′ and Z taken together form a peptide containing no more thanabout fifteen amino acids. In one aspect, the peptide formed by X, Y′and Z is linear. Preferably X and Z are each a covalent bond.Optionally, the peptide formed by X, Y′ and Z is cyclized to form aring. When cycllzed, the ring can be formed by an amide linkage, anester linkage or a disulfide linkage between two amino acids in thepeptide. When the ring is formed by an amide linkage between theN-terminus and C-terminus of the peptide, R¹ and R² together form acovalent bond between the carbonyl at the C-terminus of the peptide andthe nitrogen at the N-terminus of the peptide.

[0029] Another embodiment of the present invention is a novel compound.The compound is represented by Structural Formula II, as describedabove.

[0030] Another embodiment, of the present invention is a method oftreating an individual suffering from a disease associated withleukocyte infiltration of tissues expressing the molecule MAdCAM-1. Themethod comprises administering to the individual a therapeuticallyeffective amount of an inhibitor represented by Structural Formula (I)or an inhibitor represented by Structural Formula (II).

[0031] Compounds of the present invention are inhibitors of the bindingof MAdCAM-1 to the receptor α4β7 and are therefore useful in thetreatment of diseases such as inflammatory bowel disease with thepotential for fewer side effects in other tissues where adhesion ismediated by α4β1 integrin, for example.

[0032] The compounds of the present invention are also useful indiagnostic and research applications. For example, the compounds can beused as immunogens (e.g., when conjugated to a suitable carrier) toinduce the formation of antibodies which selectively bind MAdCAM-1 or aportion thereof. These antibodies can in turn be used to identify cellsexpressing MAdCAM-1 on their cell surface or detect MAdCAM-1 in asample. In addition, the compounds of the present invention can belabelled and used to detect α4β7 integrin and/or quantitate expressionof α4β7 integrin on the surface of cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 is a graph illustrating a titration experiment used toselect the amount of rat anti-murine k capture antibody which was usedin adhesion assays. MAdCAM-mCk fusion protein was bound to the surfaceof a well via various amounts of capture antibody, and the adhesion offluorescently labelled HUT 78 cells to fusion protein in the presence ofincreasing amounts of anti-MAdCAM-1 antibody was monitored. Reading(Y-axis) is in arbitrary fluorescence units. 50 μl of a 10 μg/ml (▪), 5μg/ml (□), 2.5 μg/ml (♦) or 1.25 μg/ml (⋄) solution of purified ratanti-murine k antibody was used. Anti-murine MAdCAM-1 antibody MECA-367was used as neat supernatant or diluted 1:2, 1:4, 1:8 or 1:16 (indicatedon the X-axis by :1 (neat), :2, :4, :8, and :16, respectively). “None”indicates no MECA-367 antibody (buffer control) was present.

[0034]FIG. 2 is a graph illustrating the inhibition by compound 793-laof the adherence of fluorescently labelled cells bearing α4β7 (HUT 78human T cell lymphoma cells) to a MAdCAM-mCk fusion protein bound bycapture antibody. The various concentrations (μM) of compound used wereplotted against a ratio (calculated from measurements of adherentfluorescent cells; see Example 2).

[0035]FIG. 3 is a schematic illustration of a MAdCAM-1/mCk fusion geneand encoded fusion protein. A BamHI site at the junction of the MAdCAM-1and mCk sequences introduces codons for Gly-Ser (SEQ ID NO: 63 and SEQID NO: 64). The fusion gene encodes the signal peptide (SEQ ID NO: 65and SEQ ID NO: 66, partial signal peptide) and complete extracellulardomain of MAdCAM-1 through the threonine residue at posItion 365. Singleletter amino acid codes are used.

[0036]FIG. 4 is an illustration of the nucleotide sequence determinedfrom subclones of cDNA clone 4 encoding human MAdCAM-1 (SEQ ID NO: 67),and the sequence of the predicted protein encoded by the open readingframe (MAdCAM-1) (SEQ ID NO: 68). The predicted signal peptide andtransmembrane region are underlined in bold. Cysteine residues of thetwo Ig-like domains are boxed, as are potential N-linked glycosylationsites. The mucin domain consisting of 71 amino acids is outlined by athin bold line. The LDTSL (SEQ ID NO: 1) motif begins at amino acid 63.

[0037]FIG. 5 is an illustration of the nucleotide sequence determinedfrom subclones of cDNA clone 20 encoding human MAdCAM-1 (SEQ ID NO: 69),and the sequence of the predicted protein encoded by the open readingframe (MAdCAM-1) (SEQ ID NO: 70). The predicted signal peptide andtransmembrane region are underlined in bold. Cysteine residues of thetwo Ig-like domains are boxed, as are potential N-linked glycosylationsites. The mucin domain consisting of 47 amino acids is outlined by athin bold line. The LDTSL (SEQ ID NO: 1) motif begins at amino acid 63.The two human cDNA clones are probably isoforms encoded by genomic DNA,generated, for example, by alternative splicing or by transcription oftwo different alleles.

DETAILED DESCRIPTION OF THE INVENTION

[0038] As used herein, “lower alkyl” refers to a hydrocarbon containingfrom one to about twelve carbon atoms. In one aspect, the term “C3-C12alkyl” is employed to include all lower alkyls except —CH3 and —CH₂—CH₃.The hydrocarbon can be saturated or can contain one or more units ofunsaturation. The hydrocarbon can also be branched, straight chained orcyclic.

[0039] A “cyclic hydrocarbon” refers to a cycloalkyl group. A“cycloalkyl group” includes carbocyclic rings (e.g., cyclopentyl andcyclohexyl) as well as carbocyclic rings in which one or more carbonatoms is replaced by a heteroatom (e.g., morpholino, piperidinyl,pyrrolidinyl, thiomorpholino or piperazinyl), Also included arecycloalkyl rings fused to other cycloalkyl rings (e.g., decalinyl) orfused to aromatic or heteroaromatic groups (e.g., indanyl, tetralinyland 9-xanthenyl). A “cyclic hydrocarbon” also includes bridgedpolycyclic structures such as adamantyl and nonbornyl. As with otherlower alkyl groups, a cyclic hydrocarbon can be substituted.

[0040] As used herein, “aryl” groups are defined to include aromaticring systems such as phenyl. “Heteroaryl” groups are defined to includearomatic ring systems which contain heteroatoms, such as 2-furanyl,3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 2-pyranyl and 3-pyranyl.“Aryl” and “heteroaryl” groups also include fused polycyclic aromaticring systems in which the aryl or heteroaryl ring is fused to one ormore other aryl, heteroaryl or cycloalkyl rings. Examples includea-naphthyl, β-naphthyl, 1-anthracenyl, 2-anthracenyl, 2-benzothienyl,3-benzothienyl, 2-benzofuranyl, 3-benzofuranyl, 2-indolyl, 2-quinolinyl,3-quinolinyl, acridinyl and 9-thioxanthanyl. Also included arepolycyclic aromatic ring systems in which aryl groups, heteroaryl groupsor an aryl and a heteroaryl group are connected by a covalent bond(e.g., 4-phenylphenyl, 3,5-diphenylphenyl, 4-(2-thienyl)phenyl and4-(2-furanyl)phenyl)) or a —(CH)_(n)— bridge (e.g.,4-(phenyl-CH₂)phenyl, 4-(phenylCH₂CH₂-) phenyl, 4-(-CH₂-2-thienyl)phenyland 4-(-CH₂CH₂-2-thienyl)phenyl), wherein n is an integer from 1 toabout 5.

[0041] Suitable subscituents on a lower alkyl group, an aryl group or aheteroaryl group include C1-C2 alkoxy, ketone, aldehyde, (lower alkyl)ester, aryl, substituted aryl, heteroaryl, substituted heteroarylbenzyl, lower alkyl, fluoro, bromo, iodo, cyano, nitro and the like. Alower alkyl group, an aryl group or a heteroaryl group may have morethan one substituent (e.g., 2,2,3,3-tetramethylcyclopropyl,3,5-diphenylphenyl and 2,4-dichlorophenyl), 2-bromo-4-nitro-pentyl and2-(3,5-dibromo-benzofuranyl). In addition, a substituted lower alkylgroup can have multiple substituents on one carbon atom (e.g.,diphenylmethyl, triphenylmethyl, 2,2,3,3-tetramethylcyclopropyl andtrifluoromethyl).

[0042] R¹ and R² are groups covalently bonded to the N-terminal and theC-terminal, respectively, of the peptide sequences of the compoundsrepresented by Structural Formulas (I) and (II).

[0043] In one preferred embodiment, R³ is selected from the groupconsisting of adamantyl, adamantylmethyl, a C1-C4 alkyl group, a C3-C7cycloalkyl group, a C3-C7 cycloalkyl group substituted with a C1-C4alkyl group, an aryl group substituted with a C3-C7 cycloalkyl group,phenyl substituted with a C1-C8 alkyl group, an aryl group,2-anthracenyl, diphenylmethyl, 1-napthyl, 2-naphthyl, benzyl, indanyl,tetralinyl, triphenylmethyl, triphenyl (C1-C4 alkyl), 9-fluorenyl,styryl, a heteroaryl group, furanyl, thienyl, 9-xanthanyl,9-thioxanthanyl, acridinyl, pyridyl, quinolinyl, a C1-C4 alkyl groupsubstituted with an aryl group (e.g.,, benzyl and phenylethyl) and aC1-C4 alkyl group substituted with heteroaryl (furanylmethyl,thienylmethyl, pyridylmethyl, quinolinylmethyl). More preferably, R³ isselected from the group consisting of triphenylmethyl, diphenylmethyl,3,5-diphenylphenyl, 2-furanyl, 3-furanyl, 9-xanthenemethyl,2,2,2-triphenylethyl, 2-anthracene, methyl, cyclopentyl, 2-indolyl,2-indanyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl,3-benzothienyl, cyclohexyl, 5-phenylpentyl,4-isobutyl-α-methylphenylmethyl, 4-biphenylmethyl, α-naphthylmethyl,4-heptylphenyl, phenylmethyl, trans 2-phenylethenyl and2,2,3,3-tetramethylcyclopropyl.

[0044] In another preferred embodiment, R³ is a substituted orunsubstituted aryl or heteroaryl group. Examples include monocyclic andbicyclic nitrogen-containing heteroaromatic groups, such as a quinolinylgroup (e.g., 2-quinolinyl, 2-phenyl-4-quinolinyl, 3-quinolinyl,4-quinolinyl, 7-quinolinyl), an isoquinolinyl group (e.g.,3-isoquinolinyl), an indolyl group (e.g., 2-indolyl and5-chloro-2-indolyl), a quinoxalinyl group (e.g., 2-quinoxalinyl), acinnolinyl group, and a pyrazinyl group. Also included are vinyl groupssubstituted with substituted or unsubstituted aryl or heteroaryl groups(e.g., cis and trans styryl and stilbyl, (3-pyridyl) —CH═CH—),polycarbocyclic aromatic hydrocarbons (e.g., 2-naphthyl and 2-anthracyl)and oxygen-containing polycyclic aromatic hydrocarbons (e.g.,9-xanthanyl, 2-benzopyranone, 3-benzopyranone and 2-benzofuranyl).Suitable substituents for an aryl or heteroaryl group are as describedabove.

[0045] R⁴ and R⁵ are preferably independently selected from the groupconsisting of a C1-C5 alkyl group, a C1-C5 alkyl group substituted withC1-C4 alkoxy, a C3-C7 cycloalkyl group, a C3-C7 cycloalkyl groupsubstituted with a C1-C5 alkyl group, an aryl group, a substituted C1-C5alkyl group (e.g.,, suitable substituents include a hydroxyl group,phenyl, substituted phenyl (e.g., suitable substituents include a C1-C5alkyl group, C1-C4 alkoxy, halogen, nitro and trifluoromethyl)), a C1-C4alkyl group substituted with an aryl group (e.g., benzyl, phenylethyl,diphenylethyl, 9-fluorenyl), a heteroaryl group (e.g., benzofuranyl,benzothienyl, furanyl, pyridiryl, auinolinyl, and thienyl) and a C1-C5alkyl group substituted with a heteroaryl group (e.g., furanylmethyl,thienylmethyl, pyridylmethyl and quinolinylmethyl).

[0046] In addition, R⁴ and R⁵ may, taken together, form a heterocyclicring including piperidinyl, pyrrolidinyl, morpholino, thiomorpholino orpiperazlnyl where the N⁴-position of the piperazine ring is substitutedby a group consisting of hydrogen, acetyl, benzoyl, alkyl, benzyl orphenyl. More preferably, R⁴ and R⁵ are eacn independently selected fromthe group consisting of 2-hydroxyethyl, benzyl, 2-benzofuranyl,3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, —CH₂-2-thienyl,—CH₂-3-thienyl, —CH₂-2-furanyl, —CH₂-3-furanyl, 3,4-dimethoxybenzyl, andisopentyl.

[0047] Examples of compounds of the present invention include thefollowing:

[0048] Cpc-NH-Asp-Thr-N(CH₂CH₂OH) (Benzyl)

[0049] Cpc-NH-Asp-Thr-NH-Hexyl

[0050] Cpc-NH-Asp-Thr-NH-(3,4-Dimethoxybenzyl)

[0051] Cpc-NH-Asp-Thr-NH-CH(Phenyl)₂

[0052] Idc-NH-Asp-Thr-NH-Benzyl

[0053] Chc-NH-Asp-Thr-NH-CH₂Thienyl

[0054] Bpc-NH-Asp-Thr-NH-Benzyl

[0055] Indc-NH-Asp-Thr-NH-Isopentyl

[0056] Bpc-NH-Asp-Thr-NH-CH₂Thienyl

[0057] Abbreviations are defined in Table 3.

[0058] In a preferred embodiment, R³ is selected from the groupconsisting of diphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl,2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and3-benzothienyl, R⁴ is selected from the group consisting of2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl,3-benzothienyl, —CH₂-2-thienyl, —CH₂-3-thienyl, —CH₂-2-furanyl,—CH₂-3-furanyl and P, is —H.

[0059] As used herein, “[AA]” represents an amino acid and “[AA]₁. . .[AA]_(n)” represents a peptide, wherein [AA]₁ is the N-terminal aminoacid and [AA]_(n) is the C-terminal amino acid. Unless otherwiseindicated, amino acids in the peptides are presented from left to right,with the left end being the N-terminus and the right end being theC-terminus. The amino acids within the peptide can be similarlydesignated. For example, “[AA]₂” and “[AA]_(n-1)” refer to the secondamino acid from the N-terminal and C-terminal, respectively. Thisnomenclature permits each amino acid of X and Z to be designated. Forexample,, “[AA]₁ of Z” refers to the amino acid at the N-terminus of Zwhen Z is a peptide. “[AA]₂ of X” refers to the second amino acid fromthe N-terminus of X when X is a peptide. “[AA]_(n-1) of X” refers to thesecond amino acid from the C-terminus of X when X is a peptide. It is tobe understood that the amino acids in Y are numbered in sequence from1-5 (Y′ is numbered in sequence from 1-5, 1-3 or 1-2 when Y′ is apentapeptide, a tripeptide or a dipeptide, respectively) beginning atthe N-terminus and ending at the C-terminus. Thus, “[AA]₂ of Y” refersto the second amino acid from the N-terminal of Y. It is also to beunderstood that “[AA]₁ of X” (or “[Aa]₁ of Z”) can be used to representX (or Z) when X (or Z) is a single amino acid.

[0060] In one embodiment Y is Y′, e.g., Asp-Thr, Leu-Asp-Thr or apentapeptide [Aa₁-[AA]₂-[AA]₃-[AA]₄-[AA]₅ having the sequenceLeu-Asp-Thr-Ser-Leu (SEQ ID NO: 1) with the proviso that a single one of[AA]₁, [AA]₂, [AA]₃, [AA]₄ or [AA]₅ can vary, being any naturallyoccurring amino acid. For example, the pentapeptide can beXaa-Asp-Thr-Ser-Leu (SEQ ID NO.: 85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO:86), Leu-Asp-Xaa-Ser-Leu (SEQ ID NO: 87), Leu-Asp-Thr-Xaa-Leu (SEQ IDNO: 88), or Leu-Asp-Thr-Ser-Xaa (SEQ ID NO: 89). A “naturally occurringamino acid” is an amino acid that occurs in nature. “Naturally occurringamino acids” includes, but are not limited to serine, threonine,cysteine, glycine; valine, alanine, leucine, isoleucine, aspartic acid,glutamic acid, glutamine, asparagine, lvsine, arginine, ornithine,tyrosine, phenylalanine, histidine, proline, 4-hydroxyproline,tryptophan and methionine.

[0061] For Structural Formula (I), if X is an amino acid, X may not bearginine or a derivative of arginine. In addition, for StructuralFormula (I) if X is a peptide, the amino acid at the C-terminus of X maynot be arginine or an arginine derivative. Arginine derivatives includeN and/or N′ C1-C4 alkylated arginines (e.g., N-alkyl arginine,N,N-dialkyl arginine, N,N′-dialkyl arginine, and N,N,N′-trialkylarginine). Arginine derivatives also include arginine mimics (e.g.,p-aminomethyl arginine), arginine isomers (e.g., norarginine), argininehaving substituents in the side chain (e.g., nitro, halo or C1-C4 alkyl)and arginines containing one or more additional carbon atoms in the sidechain (e.g., homoarginine).

[0062] In a preferred embodiment, Y′ is the tripeptide Leu-Asp-Thr orthe dipeptide Asp-Thr, and examples of preferred groups for R³ includesubstituted and unsubstituted phenyl (e.g., −2,5-diCF₃, 2-CF₃, 3-CF₃,1,2-difluoro, 3,5-difluoro, 2,5,6-trifluoro, 3-(n-hexyloxy), 3-chloro,4-t-butyl and 4-phenyl substituted phenyl), thienyl (e.g., 2-thienyl and3-halo-2-thienyl), indolyl (e.g., 2-indolyl), pyrimidyl (e.g.,1-chlor-3-trifluoro-4-pyrimidyl), pyridyl (e.g.,2-methyl-5-chloro-3-pyridyl), benzofuranyl (e.g., 2-benzofuranyl),isoqzuinlinyl (e.g., 3-isoquinolinyl), 3-isoquinolinyl-CO—NH—(CH₂)_(x)—(wherein x is an integer from 1-4) and benzopyranone groups (e.g., 2-and 3-benzopyranone). R³ is more preferably 3-isoquinolinyl or2-benzoluranyl. R⁴ is preferably —H and R⁵ is preferably benzyl;substituted benzyl (e.g., 3-CF₃, 3-methyl, 4-CH₃, 2-CH₃, 4-fluoro,3,4-difluoro, 2,5-difluoro, 3,4-methylenedioxy, 4-N-morpholino and4-OCH₃ substituted benzyl), phenethyl, substituted phenethyl (e.g.,3,4-dimethoxy and 4-SO₂NH₂ substituted phenethyl), phenpropyl,substituted phenpropyl, heteroaryl-CH₂— (wherein heteroaryl is, e.g.,3-furanyl, 3-thienyl and 4-pyridinyl), substituted heteroaryl-CH₂—(e.g., 6-(2-methyl)-quinolinyl), lower alkyl, substituted lower alkyl(e.g., isopropyl, methoxymethyl, 2-hydroxyethyl, 3-hydroxypropyl,3-(2-methyl-N-piperidinyl)propyl, 1-(ethyl)-1-propyl,2-(1-cyclohexene)ethyl and 1-(cyclohexyl)eethyl) and cycloalkyl (e.g.,cyclopropyl, cyclopentyl, cyclohexyl and cyclooctyl). Other examples ofR⁵ are represented by the following structural formulas:

[0063] In addition, taken together, R₄ and R₅ can form a heterocyclicring such as pyrrolidinyl, substituted pyrrolidinyl (e.g., 3,3-dimethylsubstituted and 3-dimethylamino substituted pyrrolidinyl), indoline,isomers of indoline, substituted indoline, substituted isomers ofindoline, tetrahydroisoquinoline, substituted tetrahydroisoquinoline,tetrahydroquinoline, substituted tetrahydroquinoline, piperidones (e.g.,4-piperidone), substituted piperidones, piperidine, substitutedpiperidines, tetrahydro-oxazines (e.g., 1,3-tetrahydro-oxazine andmorpholine) and substituted tetrahydro-oxazines (e.g.,2,4-dimethyl-1,3-tetrahydro-oxazine and 3,5-dimethyl morpholine).

[0064] In another embodiment, when Y′ is the tripeptide Leu-Asp-Thr orthe dipeptide Asp-Thr, R¹ is represented by Structural Formula (IV):

[0065] A is an aryl group, a substituted aryl group a heteroaryl groupor a substituted heteroaryl group, as described above. Examples include3-isoquinolinyl, 2-indolyl, 5-chloro-2-indolyl, 2-benzofuranyl, phenyland trans-styryl. n and m are each independently zero or one.Preferably, n+m=1.

[0066] The substitution pattern on the phenyl ring in Structural Formula(IV) can be ortho, meta or para.

[0067] In another preferred embodiment Y or Y′ is the pentapeptide[AA]-[AA]₂-[AA]₃-[AA]₄-[AA]₅] wherein:

[0068] amino acid [AA]₁ is selected from the group consisting ofleucine, isoleucine, alanine, valine, glycine, phenylalanine andN-methylleucine;

[0069] amino acid [AA]₂ is selected from the group consisting ofaspartic acid, glutamic acid, phenylalanine and tyrosine;

[0070] amino acid [AA]₃ is selected from the group consisting ofthreonine, serine, valine, proline and 4-hydroxyoroline;

[0071] amino acid [AA]₄ is selected from the group consisting of serine,cysteine and threonine; and

[0072] amino acid [AA]₅ is selected from the group consisting ofleucine, alanine, valine, isoleucine, alanine, glycine, phenylalanineand N-methylleucine.

[0073] In another aspect, any one or more of [AA]₁, [AA]₂, [AA]₃, [AA]₄and [AA]₅ in the pentapeptide of Y or Y′ can also be a non-naturallyoccurring amino acid. [AA]₁ and/or [AA]₅ can be a non-naturallyoccurring amino acid in which the side chain is a C2-C7 substituted orunsubstituted alkyl group or a substituted phenyl group. Side chainalkyl groups can be straight chained, branched or cyclic. Suitablesubstituents for an alkyl side chain include non-polar groups such asC1-C2 alkyl, halo, cyano, C1-C3 alkoxy, phenyl or substituted phenyl.Suitable substituents for a side chain phenyl group include non-polargroups such as C1-C2 alkyl, halo, cyano or —C1-C3 alkoxy. [AA]₂ can be anon-naturally occurring amino acid having a C3-C6 straight chained orbranched alkyl group substituted with a carboxylic acid. [AA]₃ and/or[AA]₄ can be a non-naturally occurring amino acid in which the sidechain is a C2-C6 straight chained or branched alkyl group substitutedwith an alcohol or thiol.

[0074] The compounds represented by Structural Formulas (I) and (II) arepeptides X-Y-Z or X-Y-Z, respectively, with groups attached to theN-terminus and C-terminus. Preferably, X and Z are covalent bonds, i.e.the compounds represented by Structural Formulas (I) and (II) arepeptides Y or Y′, respectively, with group R attached to the N-terminalof Y or Y′, and with group R2 attached to the C-terminal of Y or Y′. Inone aspect, X-Y-Z or X-Y′-Z is a linear peptide. In another aspect,X-Y-Z or X-Y′-Z is cyclized.

[0075] As used herein, “cyclized” refers to forming a ring by a covalentbond between suitable side chains of two amino acids in the peptide. Forexample, a disulfide can be formed between the sulfur atoms in the sidechains of two cysteines. Alternatively, an ester can be formed betweenthe carbonyl carbon in the side chain of, for example, glutamic acid oraspartic acid, and the oxygen atom in the side chain of, for example,serine or threonine. An amide can be formed between the carbonyl carbonin the side chain of, for example, glutamic acid or aspartic acid, andthe amino nitrogen in side chain of, for example, lysine or ornithine.

[0076] “Cyclized” also refers to forming a ring by a peptide bondbetween the nitrogen atom at the N-terminus and the carbonyl carbon atthe C-terminus. In this case, R¹ and R² together form a covalent bond.

[0077] “Cyclized” also refers to forming a ring by forming a covalentbond between the nitrogen at the N-terminus of the compound and the sidechain of a suitable amino acid in the peptide. For example, an amide canbe formed between the nitrogen atom at the N-terminus and the carbonylcarbon in the side chain of aspartic acid or glutamic acid.Alternatively, the compounds represented by Structural Formulas (I) and(II) can also be cyclized by forming a covalent bond between thecarbonyl at the C-terminus of the compound and the side chain of asuitable amino acid in the peptide. For example, an amide can be formedbetween the carbonyl carbon at the C-terminus and the amino nitrogenatom in the side chain of lysine or ornithine; an ester can be formedbetween the carbonyl carbon at the C-terminus and the hydroxyl oxygenatom in the side chain of serine or threonine.

[0078] Preferably, the ring of the cyclized compounds of the presentinvention contains four to nine amino acids. The peptide is preferablycyclized to maximize within the ring the number of amino acids involvedin the binding of the peptide to a ligand of MAdCAM-1, such as humanα4β7. Thus, the ring contains at least four of the five amino acids in Yor Y′, when Y or Y′ is a pentapeptide. More preferably, the ringcontains all of the amino acids in Y or Y′.

[0079] For compounds represented by Structural Formula (I), the ring canbe formed by a bond between the side chain of an amino acid selectedfrom the group consisting of [AA]_(n-3) of X, [AA]_(n-2) of X,[AA]_(n-1) of X, [AA]_(n) of X (if the specific amino acid is present inX) and [AA]₁ of Y and the side chain of any one of the amino acidsselected from the group consisting of [AA]₅ of Y, [AA]₁ of Z, [AA]₂ ofZ, [AA]₃ of Z and [AA]₄ of Z (if the specific amino acid is present in(X and/or Z), with the proviso that the ring contains from four to nineamino acids. The ring can also be formed by a bond between the sidechain of [AA]₄ and the side chains of any one of [AA]_(n-4) of X,[AA]_(n-3) of X, [Aa]_(n-2) of X, [AA]_(n-1) of X, [AA]_(n) of X (if thespecific amino acid is present in X) or [AA]₁ of Y when the ringcontains only four of the five amino acids of Y. Alternatively, the ringcan also be formed by a bond between the side chain of [AA]₂ and theside chains of any one of [AA]₅ of Y, [AA]₁ of Z, [AA]₂ of Z. [AA]₃ ofZ. [AA]₄ of Z or [AA]₅ of Z (if the specific amino acid is present in Z)when the ring contains only four of the five amino acids of Y.

[0080] For compounds represented by Structural Formula (II), when Y′ isa pentapeptide, the ring can be formed as described in the precedingparagraph. When Y′ is a tripeptide, the ring can be formed by a bondbetween the side chain functional group of an amino acid selected fromthe group consisting of [AA]_(n-5) of X, [AA]_(n-4) of X, [AA]_(n-3) ofX, [AA]_(n-2) of X, [AA]_(n-1) of X, [AA]_(n) of X and [AA]₁ of Y′ andthe side chain functional group of any one of the amino acids selectedfrom the group consisting of [AA]₃ of Y′, [AA]₁ of Z, [AA]₂ of Z, [AA]₃of Z, [AA]₄ of Z, [AA]₅ of Z and [AA]₆ of Z (if the specific amino acidis present in X and/or Z), with the proviso that the ring contains fromfour to nine amino acids. When Y′ is a dipeptide, the ring can be formedby a bond between the side chain of an amino acid selected from thegroup consisting of [AA]₂ of Y′, [AA]_(n-5) of X, [AA]_(n-4) of X,[AA]_(n-3) of X, [AA]_(n-2) of X, [AA]_(n-1) of X, [AA]_(n) of X and[AA]₁ of Y′ and the side chain functional group of any one of the aminoacids selected from the group consisting of [AA]₂ of Y′, [AA]₁ of Z,[AA]₂ of Z, [AA]₃ of Z, [AA]₄ of Z, [AA]₅ of Z, [AA]₆ of Z and [AA]₇ ofZ (if the specific amino acid is present in X and/or Z), with theproviso that the ring contains from four to nine amino acids.

[0081] When the ring contains all of the amino acids of Y or Y′, thecompound of Structural Formula I or II can be cyclized by a peptide bondbetween the nitrogen at the N-terminus of X and the carbonyl carbon atthe C-terminal of Z.

[0082] In one embodiment, X is an amino acid or has an amino acidsequence which is the same as that immediately N-terminal to the LDTSL(SEQ ID NO: 1) motif of human MAdCAM-1, and Z is an amino acid or has anamino acid sequence which is the same as that immediately C-terminal tothe LDTSL (SEQ ID NO: 1) motif of human MAdCAM-1. In a further aspect,any one or two amino acids in X and Z can replaced by a naturallyoccurring amino acid or a non-naturally occurring amino acid, asdescribed above.

[0083] Peptide sequences in the compounds of the present invention maybe synthesized by solid phase peptide synthesis (e.g., BOC or FMOC)method, by solution phase synthesis, or by other suitable technlauesincluding combinations of the foregoing methods. The BOC and FMOCmethods, which are established and widely used, are described inMerrifield, J. Am. Chem. Soc. 88:2149 (1963) Meienhofer, HorrmonalProteins and Peptides, C. H. Li, Ed., Academic Press, 1983, pp. 48-267;and Barany and Merrifield, in The Peptides, E. Gross and J. Meienhofer,Eds., Academic Press, New York, 1980, pp. 3-285. Methods of solid phasepeptide synthesis are described in Merrifield, R. B., Science, 232: 341(1986); Carpino, L. A. and Han, G. Y., J. Org. Chem., 37: 3404 (1972);and Gauspohl, H. et al., Synthesis, 5: 315 (1992)). The teachings ofthese six articles are incorporated herein by reference in theirentirety. Examples of the synthesis of the compounds having thestructure represented by Structural Formulas (I) and (II) are disclosedin Examples 1, General Procedures A-C.

[0084] N- and C-terminal modified peptides can be prepared on an oximeresin using a modification of procedures described in DeGrado andKaiser, J. Org. Chem. 47:3258 (1982), the teachings of which areincorporated herein by reference. Oxime resin can be obtained from NovaBiochem, Inc. The resin is acylated with, for example, Fmoc-Thr(t-Bu)-OH(4-6 equivalents), HBTU (4-6 equivalents)/NMM (8-12 equivalents) indimethylformamide (DMF) at room temperature over about twenty hours. Theresin is then washed with DMF, followed by methylene chloride and driedunder vacuum prior to use. A peptide can then be synthesized using theFmoc-Thr(t-Bu)-Oxime resin and General Procedure C in Example 1. Theresulting peptide is reacted with the desired amine (8-12 equivalents)in methylene chloride for twelve hours. The resin is then washed withmethylene chloride The filtrate is washed with 5% citric acid in waterand dried over anhydrous magnesium sulfate. The solvent is removed andthe resulting residue dried under vacuum to give the desired N- andC-terminal modified residue.

[0085] Methods of cyclizing compounds having peptide sequences aredescribed, for example, in Lobl et al., WO 92/00995, the teachings ofwhich are incorporated herein by reference in their entirety. Cyclizedcompounds can be prepared by protecting the side chains of the two aminoacids to be used in the ring closure with groups that can be selectiveyvremoved while all other side-chain protecting groups remain intact.Selective deprotection is best achieved by using orthogonal side-chainprotection such as allyl (OAI) (for the carboxyl group in the side chainof glutamic acid or aspartic acid, for example), allyloxy carbonyl(Aloc) (for the amino nitrogen in the side chain of lysine or ornithine,for example) or acetamidomethyl (Acm) (for the sulfhydryl of cysteine)protecting groups. OAI and Ac are easily removed by Pd° and Acm iseasily removed by iodine treatment. Examples of cyclizing a compoundrepresented by Structural Formulas (I) and (II) by forming a disulfidebond are given in Example 1 and General Procedure D.

[0086] In another example, the cyclic peptide K*LDTSLD* (SEQ ID NO: 2),cyclized between the side chains of lysine and the N-terminal asparticacid (“*” indicates a cyclizing amino acid) peptide can assembled onPAL-PEG-PS-resin using Nu-Fmoc-amino acids and t-butyl side-chainprotection as described in General Procedures A and B in Example 1,except D* and K* are incorporated in the linear chain asFmoc-Asp(OAI)-OH and Fmoc-Lys(Aloc)-OH, respectively. Allyl functionsare removed by treatment with Pd(PPH₃)₄, morphollne andtriphenylphosphine in dry THF at room temperature and cyclization isachieved with PYBOP. Finally the peptide is deblocked and removed fromthe resin as described in General Procedures A and B.

[0087] In yet another example, the head to tail cyclic peptide TSLLD(SEQ ID NO: 62) can be assembled on Fmoc-Asp(OPAC-resin)-OAI usingFmoc-amino acids and t-butyl side-chain protection, as described inGeneral Procedures A and B in Example 1. The allyl function is removedby treatment with Pd(PPh₃)₄, morpholine and triphenylphosphine in dryTHF at room temperature and cyclization is achieved with PYBOP. Finallythe peptide is deblocked and removed from the resin as described inGeneral Procedures A and P to give cyclic LDTSL peptide. Kates, S. A.,eL al., “Peptides: Design, Synthesis and Biological Activity,” Basava C,Anantharamalah GM, eds., pp. 39-58 Boston: Birkhauser.

METHODS

[0088] Peptides which mimic the conserved amino acid mocif LDTSL (SEQ IDNO: 1) and which are modified at the N- and C-termini, including thecompounds of the present invention, are useful in a method of inhibiting(e.g., reducing or preventing) the binding of a cell bearing a ligand ofMAdCAM-1 on the cell surface to MAdCAM-1 or a portion thereof (e.g., theextracellular domain). According to the method, the cell bearing aligand for MAdCAM-1 is contacted with an effective amount of an (i.e.,one or more) inhibitor (as represented by Structural Formula (I) or(II)). As used herein, an inhibitor is a compound which inhibits(reduces or prevents) the binding of MAdCAM-1 to a ligand, includingα4β7 integrin, and/or which inhibits the triggering of a cellularresponse mediated by the ligand. An effective amount can be aninhibitory amount (such as an amount sufficient to achieve inhibition ofadhesion of a cell bearing a MAdCAM-1 ligand to MAdCAM-1). Ligands forMAdCAM-1 include α4β7 integrins, such as human α4β7 integrin, and itshomologs from other species such as mice (also referred to as α4βp orLPAM-1 in mice).

[0089] For example, the adhesion of a cell which naturally expresses aligand for MAdCAM-1, such as a leukocyte (e.g., g lymphocyte, Tlymphocyte) or another cell which expresses a ligand for MAdCAM-1 (e.g.,a recombinant cell), to MAdCAM-1 can be inhibited in vivo or in vitroaccording to the method. In one embodiment, the MAdCAM-mediatedinteraction of a first cell bearing a ligand for MAdCAM-1 with a secondcell bearing MAdCAM-1 is inhibited by contacting the first cell with aninhibitor according to the method.

[0090] The adhesion of cells to MAdCAM-1 or a suitable portion thereofcan be inhibited. For example, MAdCAM-1 or a suitable portion thereofcan be a soluble protein or can be expressed on the surface of asuitable cell, such as a cell which naturally expresses MAdCAM-1 (e.g.,an endothelial cell), a suitable cell line, or another cell whichexpresses MAdCAM-1 (e.g., a recombinant cell). Suitable portions ofMAdCAM-1 include, for example, a portion comprising the LDTSL (SEQ IDNO: 1) motif capable of mediating adhesion (e.g., a portion comprisingthe entire extracellular domain, or both N-terminal immunoglobulin-likedomains) (see e.g., Briskin, et al., WO 96/24673, published Aug. 15,19961). MAdCAM-1 or a portion thereof can be part of a larger molecule,such as a fusion protein. For example, a soluble hybrid proteincomprising a mammalian (e.g., human or other primate, murine) MAdCAM-1moiety fused at its C-terminus, to the N-terminus of an immunoglobulinmoiety (e.g., one or more immunoglobulin constant regions) to obtain animmunoadhesin, such as those prepared according to Capon eL al. (U.S.Pat. No. 5,428,130), can be used. For example, as shown herein, theadhesion of a T cell lymphoma line which expresses α4β7 to a fusionprotein comprising the extracellular domain of murine MAdCAM-1 joined tothe constant region of a murine K light chain was inhibited according tothe method (See e.g., Example 2 and Table 2). The use of a fusionprotein comprising the extracellular domain of human MAdCAM-1 fused to ahuman 51 constant regions is described in Example 3. These or otherrecombinant soluble receptor molecules are useful in the method.

[0091] In another aspect, the invention relates to a method of treatingan individual (e.g., a mammal, such as a human or other primate)suffering from a disease associated with leukocyte (e.g., lymphocyte,monocyte) infiltration of tissues (including recruitment and/oraccumulation of leukocytes in tissues) which express the moleculeMAdCAM-1. The method comprises administering to the individual atherapeutically effective amount of an inhibitor (i.e., one or moreinhibitors) of Structural Formula (I) or Structural Formula (II). Forexample, inflammatory diseases, including diseases which are associatedwith leukocyte infiltration of the gastrointestinal tract (includinggut-associated endothelium), other mucosal tissues, or tissuesexpressing the molecule MAdCAM-1 (e.g., gut-assocIated tissues, such asvenules of the lamina propria of the small and large intestine; andmammary gland (e.g., lactating mammary gland)) , can be treatedaccording to the present method. Similarly, an individual suffering froma disease associated with leukocyte infiltration of tissues as a resultof binding of leukocytes to cells (e.g., endothelial cells) expressingthe molecule MAdCAM-1 can be treated according to the present invention.

[0092] Diseases which can be treated accordingly include inflammatorybowel disease (IBD), such as ulcerative colitis, Crohn's disease,ileitis, Celiac disease, nontropical Sprue, enteropathy associated withseronegative arthropathies, microscopic or collagenous colitis,eoslnophilic gastroenteritis, or pouchitis resulting afterproctocolectomy, and ileoanal anastomosis.

[0093] Pancreatitis and insulin-dependent diabetes mellitus are otherdiseases which can be treated using the present method. It has beenreported that MAdCAM-1 is expressed by some vessels in the exocrinepancreas from NOD (nonobese diabetic) mice, as well as from BALB/c andSJL mice. Expression of MAdCAM-1 was reportedly induced on endothelum ininflamed islets of the pancreas of the NOD mouse, and MAdCAM-1 was thepredominant addressin expressed by NOD islet endothelium at early stagesof insulitis (Hanninen, A., et al., J. Clin. Invest., 92: 2509-2515(1993)). Further, accumulation of lymphocytes expressing α4β7 withinislets was observed, and MAdCAM-1 was implicated in the binding oflymphoma cells via α4β7 to vessels from inflamed islets (Hanninen, A.,et al., J. Clin, Invest., 92: 2509-2515 (1993)).

[0094] Examples of inflammatory diseases associated with mucosal tissueswhich can be treated according to the present method include mastitis(mammary gland), cholecystitis, cholangitis or pericholangitis (bileduct and surrounding tissue of the liver), chronic bronchitis, chronicsinusitis, asthma, and graft versus host disease (e.g., in thegastrointestinal tract). As seen in Crohn's disease, inflammation oftenextends beyond the mucosal surface, accordingly chronic inflammatorydiseases of the lung which result in interstitial fibrosis, such ashypersensitivity pneumonitis, collagen diseases, sarcoidosls, and otheridiopathic conditions can be amenable to treatment.

[0095] Some studies have suggested that the cell adhesion molecule,ICAM-1, can mediate leukocyte recruitment to inflammatory sites throughadhesion to leukocyte surface ligands, i.e. , Mac-1, LFA-1 or 94 l(Springer, Nature, 346:425-434 (1990)). In addition, vascular celladhesion molecule-l (VCAM-1), which recognizes the α4β1 integrin(VLA-4), has been reported to play a role in in vivo leukocyterecruitment (Silber et al., J. Clin. Invest. 93:1554-1563 (1994)). Ithas been proposed that IBD can be treated by blocking the interaction ofICAM-1 with LFA-1 or Mac-1, or of VCAM-1 with α4β1 (e.g., WO 93/15764).However, these therapeutic targets are likely to be involved ininflammatory processes in multiple organs, and a functional blockadecould cause systemic immune dysfunction. in contrast to VCAM-1 andICAM-1, MAdCAM-1 is preferentially expressed in the gastrointestinaltract and mucosal tissues, binds the α4β7 integrin found on lymphocytes,and participates in the homing of these cells to mucosal sites, such asPeyer's patches in the intestinal wall (Hamann et al., J. Immunol.,152:3282-3293 (1994)). As inhibitors of the binding of MAdCAM-1 to α4β7integrin, the compounds of the present invention have the potential forfewer side effects due to e.g., effects on other tissue types whereadhesion is mediated by other receptors, such as α4β1 integrin.

[0096] According to the method, an inhibitor can be administered to anindividual (e.g., a human) alone or in conjunction with another agent,such as an additional pharmacologically active agent (e.g.,sulfasalazine, an antiinflammatory compound, or a steroidal or othernon-steroidal antiinflammatory compound). A compound can be administeredbefore, along with or subsequent to administration of the additionalagent, in amounts sufficient to reduce or prevent MAdCAM-mediatedbinding to a ligand for YAdCAM-1, such as human α4β7.

[0097] An effective amount of an inhibitor can be administered by anappropriate route in a single dose or multiple doses. An effectiveamount is a therapeutically effective amount sufficient to achieve thedesired therapeutic and/or prophylactic effect (such as an amountsufficient to reduce or prevent MAdCAM-mediated binding to a MAdCAMligand, thereby inhibiting leukocyte adhesion and infiltration andassociated cellular responses. Suitable dosages of inhibitors ofStructural Formula (I) or (II) for use in therapy, diagnosis orprophylaxis, can be determined by methods known in the art and can bedependent, for example, upon the individual's age, sensitivity,tolerance and overall well-being. For example, dosages can be from about0.1 mg/kg to about 50 mg,/kg body weight per treatment.

[0098] A variety of routes of administration are possible including, butnot necessarily limited to parenteral (e.g., intravenous, intraarterial,intramuscular, subcutaneous injection), oral (e.g., dietary), topical,inhalation (e.g., intrabronchial, intranasal or oral inhalation,intranasal drops), or rectal, depending on the disease or condition tobe treated. Oral and parenteral administration are preferred modes ofadministration.

[0099] Formulation of an inhibitor to be administered will varyaccording to the route of administration selected (e.g., solution,emulsion, capsule). An appropriate composition comprising the compoundto be administered can be prepared in a physiologically acceptablevehicle or carrier. For solutions or emulsions, suitable carriersinclude, for example, aqueous or alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Parenteral vehiclescan include sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's or fixed oils. Intravenous vehiclescan include various additives, preservatives, or fluid, nutrient orelectrolyte replenishers (See, generally, Remington's PharmaceuticalScience, 16th Edition, Mack, Ed. (1980)). For inhalation, the compoundcan be solubilized and loaded into a suitable dispenser foradministration (e.g., an atomizer, nebulizer or pressurized aerosoldispenser).

[0100] The present method can be used to assess the inhibitory effect ofa compound of the present invention and of other potential antagonistsuseful in the method on the interaction of MAdCAM-1 with a ligand forMAdCAM-1 in vitro or in vivo. For example, compounds of the presentinvention were assayed for their ability to inhibit MAdCAM-1 binding tohuman α4β7 integrin using an adhesion assay described in Example 2 andExample 3. Other suitable assays can be used to assess the ability ofcompounds to inhibit binding of MAdCAM-1 to a ligand for MAdCAM-1. Forexample, other fusion proteins (e.g., a chimeric protein or“immunoadhesin”) can be constructed and used in an assay such as theassay described in Example 2 or other suitable assays. A fusion proteincomprising human MAdCAM-1 or a portion thereof (e.g., the entireextracellular domain or the two N-terminal immunoglobulin domains)joined to an immunoglobulin heavy or light chain constant region can beproduced and used in an assay similar to that described in Example 2,using a capture antibody suitable for the immunoglobulin constant regionselected. In a different assay, unlabeled cells bearing a MAdCAM-1ligand can be contacted with such a fusion protein under conditionssuitable for binding to the ligand in the presence or absence ofcompound, and the amount of bound chimeric protein determined (e.g.,cells can be removed and the amount of chimeric protein bound determinedby, flow cytometry or other suitable methods)

[0101] Compounds suitable for use in therapy can also be evaluated invivo, using suitable animal models. Suitable animal models ofinflammation have been described. For example, NOD mice provide ananimal model of insulin-dependent diabetes mellitus. CD45 RB^(H)-/SCIDmodel provides a model in mice with similarity to both Crohn's diseaseand ulcerative colitis (Powrie, F. et al., Immunity, 1: 553-562 (1994)).Captive cotton-top tamarins, a New World nonhman primate species,develop spontaneous, often chronic, colitis that clinically andhistolgocially resembles ulcerative colitis in humans (Madara, J. L. etal., Gastroenterology, 88: 13-19 (1985)) The tamarin model and otheranimal models of gastrointestinal inflammation using BALB/c mice (a(DSS)-induced inflammation model; DSS, dextran sodium sulfate) andcommon marmosets are described in Briskin et al., U.S. Ser. No.08/523,004, filed Sep. 1, 1995, the teachings of which are incorporatedherein by reference in their entirety (see also Briskin et al., WO96/24673, published Aug. 15, 1996). Knockout mice which developintestinal lesions similar to those of human inflammatory bowel diseasehave also been described (Strober, W. and Ehrhardt, R..O., Cell, 75:203-205 (1993)).

[0102] Preferably, selective inhibition of the interaction of MAdCAM-1with a ligand thereof is achieved. Selective inhibition can be assessed,for example, by further evaluating the effect of compounds on adhesionbetween one or more other receptor-ligand pairs. For example, adhesionassays which assess the interaction of a particular receptor and ligandpair, such as (a) VCAM-1 and α4β1, (b) ICAM-1 and LFA-1, (c) fibronectinand α5β1, and (d) fibronectin and α4β1, can be conducted. Nonlimitingexamples of suitable cell lines for such adhesion assays include (a)HAMOS cells, (b) JY cells, (c) K562 cells, and (d) RAMOS cells,respectively. In these assays, isolated and/or recombinant fibronectin(Sigma Chemical Co., St. Louis, Mo.), VCAM-1, ICAM-1, or fusion proteinscomprising the ligand binding domain(s) of VCAM-1 or ICAM-1, can beused, for example.

[0103] The compounds of the present invention can also be used asimmunogens to produce antibodies, including monoclonal and polyclonalantibodies, against MAdCAM-1 using methods known in the art or othersuitable methods (see e.g., Kohler et al., Nature, 256:495-497 (1975);Galfre, G., et al., Nature, 299:550-552 (1977); Harlow et al., 1988,Antibodies: A Laboratory Manual, (Cold Spring Harbor, N.Y.); or CurrentProtocols in Molecular Biology, Vol. 2 (Supplement 27, Summer '94),Ausubel et al., Eds. (John Wiley & Sons: New York, N.Y.), Chapter 11(1991)). Antibodies can be raised against an appropriate immunogen in asuitable mammal (e.g., a mouse, rat, rabbit or sheep) For example, acompound represented by Structural Formulas (I) and (II) or a variantthereof can be produced and used as an immunogen to raise antibodies ina suitable immunization protocol.

[0104] Antibody-producing cells (e.g., a lymphocyte) can be isolatedfrom, for example, the lymph nodes or spleen of an immunized animal. Thecells can then be fused to a suitable immortalized cell (e.g., a myelomacell line), thereby forming a hybridoma. Fused cells can be isolatedemploying selective culturing techniques. Cells which produce antibodieswith the desired specificity can be selected by a suitable assay (e.g.,ELISA).

[0105] Antibodies and monoclonal antibodies produced as described have avariety of uses. For example, those against or reactive with MAdCAM-1,and preferably which bind specifically to MAdCAM-1, can be used toidentify and/or sort cells exhibiting MAdCAM-1 on the cell surface(e.g., in fluorescence activated cell sorting, histological analyses)Monoclonal antibodies specific for MAdCAM-1 can also be used to detectand/or quantitate MAdCAM-1 expressed on the surface of a cell or presentin a sample (e.g., in an ELISA). Antibodies reactive with the immunogenare also useful. For example, they can be used to detect and/orquantitate immunogen in a sample, or to purify immunogen (e.g., byimmunoaffinity purification).

[0106] This invention further relates to the diagnostic use or researchuse of antagonists of MAdCAM in the detection and/or quantitation ofα4β7 integrin present on leukocytes using a suitable label or indicator.For example, hydrocarbon fluorescence indicators such as a pyrenemoiety, or radioisotope labels such as ¹²⁵I can be attached to the arylring of the N-terminus functional group of the inhibitor or antagonistpeptide of this invention. The fluorescence properties of pyrene ringderivatives have been reported (I. A. Prokhorenko, et al., Bioorg. Med.Chem. Lett., 5:2081 (1995)). The use of labeled peptides for identifyingcells having a membrane-bound protein is described in Riper et al., J.Exp. Med. 177:851 (1993).

[0107] This diagnostic tool would be valuable in the identification ofα4β7-positive leukocytes from subjects with suspected inflammatory boweldiseases and the like. Antagonist molecules of this invention possessingthis indicator grouping are described as examples of the invention withadhesion antagonist properties.

EXEMPLIFICATION

[0108] The present invention will now be illustrated by the followingExamples, which are not intended to be limiting in any way.

Example 1: Synthesis

[0109] The novel compounds of this invention can be synthesizedaccording to the general procedures of synthesis, A-D, utilizingsolid-phase peptide synthesis methodology described herein, which areknown to person skilled in the art, or other suitable techniques. Seee.g., Merrifield, R. B., Science, 232: 341 (1980); Carpino, L. A., Han,G. Y., J. Org. Chem., 37: 3404 (1972); Gauspohl, H., et al., Synthesis,5: 315 (1992)).

[0110] For multiple peptlde synthesis the Fmoc/t-Bu protocol was used.In situ activation of the amino acid derivative was performed bybenzotriazolyl-N-oxytripyrrolidinophosphoniumhexaflurophosphate (PYBOP)or 2(1-benzotriazolyl-1-yl)-1,1,3,3-tetramethyluronium (HBTU) andN-methylmorpholine (NMM) in dimethylformamide (DMF) as a solvent. Inorder to improve the coupling efficiency and quality of the finalpeptides, each coupling reaction was carried out twice. As a solidsupport Rink Amide Am Resin(4-[2′-4-dimethoxyphenyl-Fmoc-aminomethyl]-phenoxyacetamido-norleucylaminoethylresin) was used due to its high loading and excellent swellingproperties. Fmoc group was removed by 10% piperidine and 2% DBU in DMF.The peptides were cleaved from the resin and the side chain protectinggroups were simultaneously removed by a trifluoroacetic acid (TFA)cocktail. All peptides reported in Table 2 below are carboxy-terminusblocked as the carboxamide. More details of the peptide synthesisprotocol are given below.

[0111] HPLC and Mass Spectral Analysis

[0112] All of the crude peptides were analyzed by reversed phase HPLCusing DELTAPAK C18, 5 μm column, eluted with a linear gradient of 0.1%TFA in CH₃CN/water (100% CH₃CN/0% water) to 0.1% TPA in CH₃CN/water (0%CH₃CN/100% water) over a 30 minute period with flow rate of 1 ml/minute.The purity of the samples were determined and were essentially found tocontain one component. This was confirmed by matrix-assisted laserdesorption ionization time of flight mass spectral analysis (MALDI-TOF,Kratos, Inc.) internally referenced to leucine-enkephalin and sinapinicacid. Generally, the peptides gave the mass within 1%, of MH+ or M+Na+or within experimental error of the calculated value.

[0113] General Procedure A, Peptide Synthesis

[0114] These peptides were synthesized via Fmoc/t-Butyl chemistry on aGilson 421 automated multiple peptide synthesizer starting withFmoc-Am-PS (100 mg, 0.5 mmol/g) resin. Acylations were carried out twicewith the Fmoc-amino acid (5 equivalents) , HDTU (5 equivalents) /NMM (10equivalents) using 20-45 minute coupling time. Fmoc deprotection wascarried out with 20% piperidine in DMF for 24 minutes. Treatment withReagent-R (TFA-EDT-thioanisole-anisole, 90:5:3:2) for 2 hours was usedto deblock and remove the peptides from the resin. The peptides werethen precipitated from ether and lyophilized from acetic acid.

[0115] General Procedure B, Peptide Synthesis

[0116] Peptides were synthesized via Fmoc/t-Butyl chemistry on a Gilson421 automated multiple peptide synthesizer starting with Fmoc-Am-PS (50mg, 0.5 mmol/g) resin. Acylations were carried out twice with theFmoc-amino acid (10 equivalents), HBTU (10 equivalents)/NMM (20equivalents) or PYBOP (10 equivalents)/NMM (20 equivalents) using a20-45 minute coupling time. Fmoc deprotection was carried out with 2%1,8-diazobicyclo [5,4.0]undec-7-ene (DBU) and 10% piperidine in DMF for24 minutes. Treatment with Reagent-R (TFA-EDT-thioanlsole-anisole,90:5:3:2) for 2 hours was used to deblock and remove the peptides fromthe resin. The peptides were then precipitated from ether andlyophilized from acetic acid.

[0117] General Procedure C, Synthesis of N-Acylated Peptides

[0118] The peptides were synthesized via Fmoc/t-Butyl chemistry on aGison 421 automated multiple peptide synthesizer starting withFmoc-Am-PS (50 mg, 0.5 mmol/g) resin. Acylations were carried out twicewith the Fmoc-amino acid (10 equivalents), HETU (10 equivalents)/NMM orPYBOP (10 equivalents)/NMM (20 equivalents) using 20-45 minute couplingtime. For the final acylation the Fmoc-amino acid was substituted withan appropriate organic acid. Fmoc deprotection was carried out with 20DBU and 10% piperidine in DMF for 24 minutes. Treatment with Reagent-R(TFA-EDT-thioanisole-anisole, 90:5:3:2) for 2 hours was used to deblockand remove the peptides from the resin. The peptides were thenprecipitated from ether and lyophilized from acetic acid.

[0119] General Procedure D, Synthesis Peptides Cyclized Through ADisulfide Bond

[0120] The linear acyclic peptides were synthesized as described abovein the General Procedure B. The acetamidomethyl (Acm) protecting groupfrom the cysteine side chain was simultaneously removed and peptideswere cyclized by iodine treatment. A solution of 25 mg of iodine in 5 mLof 80% aqueous acetic acid was added to 5 mg of peptide. The mixture wasshaken at room temperature for 2 hours, diluted with water (25 ml),extracted with chloroform (3×25 ml) and finally lyophilized to give thecyclized peptide.

Example 2: Biological Activity

[0121] The compounds of the present invention were evaluated for theirbiological properties. Table 2 lists a number of compounds of thepresent invention, their physical characterization, and their ability toinhibit the adhesion of α4β7-bearing cells (HUT 78 cells) to MAdCAM-1(described as percent inhibition at the concentration noted or by thecorresponding IC₅₀ value (am) determined using the adhesion assaydescribed below. IC₅₀ corresponds to the concentration of compound whichinhibits 50% of the total number of cells adhering to MAdCAM-1 in acontrol conducted in the absence of inhibitor.

[0122] Overview

[0123] A soluble fusion protein comprising murine MAdCAM-1 was producedin a baculovirus expression system and used in the adhesion assay. Afusion gene in which sequences encoding the signal peptide andextracellular domain of murine MAdCAM-1 were fused to sequences encodinga constant region of the murine kappa light chain (mC_(k)) wasconstructed, and cloned into a baculovirus shuttle vector. Fusionprotein produced from the resulting construct contained the integrinbinding sequences of murine MAdCAM-1 at the amino-terminus, and the mCksequence at the carboxy-terminus. Recombinant baculovirus encoding thefusion protein was harvested from infected Sf9 insect cells. Fusionprotein was detected in supernatants of infected Sf9 cells by ELISAassay, using a horseradish peroxidase-linked polyclonal anti-mCkantibody and chromogenic substrate according to standard protocols.Recombinant protein was also verified by immunoprecipitation withanti-murine MAdCAM-1 monoclonal antibody (MAb MECA-367).

[0124] For adhesion assays, a dose response curve obtained usingincreasing amounts of rat anti-mCk MAb indicated the amount of ratanti-mCk MAb to be used as capture antibody in the assay. Subsequently,an IC50 for Ac-LDTSL-NH2 was determined to be 278 μM.

[0125] Human T-cell lymphoma cells (HUT 78 cells) activated with Mn⁺²were used for the adhesion assay in a 96-well format. HUT 78 cells werelabelled by preincubation with ECECF-AM stain (Molecular Probes). Assayswere conducted in a final volume of 200 μl. The adhesion of HUT 78 cellsto MAdCAM-1/mCk fusion protein bound to wells via rat anti-mCk captureantibody was assessed in the presence or absence of each compound.Adhesion of HUT 78 cells was monitored using a fluorescent plate readerat a setting gain of 10 at 485/535 nM. Percent inhibition and IC₅₀values were determined.

[0126] Production of MAdCAM-1/mCk Fusion Protein Antibodies, Cells andViruses

[0127] Affinity-purifled polyclonal goat-anti-mouse-C-kappa (mC_(k))antibodies (#M33100), horseradish peroxidase-linked goat anti-mC_(k)antibodies (#M33107), and alkaline phosphatase-linked swine anti-goat H& L chains (#G50008) were purchased from Caltag. Sepharose-linked ratanti-mC_(k) affinity matrix was obtained from Zymed.

[0128] Affinity purified rat-anti-mC_(k) monoclonal antibodies wereprepared by Dr. Hans Peter Kocher (BTC) from the rat hybridoma cell line187.1 (American Type Culture Collection, 10801 University Blvd.,Manassas, Va. 20110-2209, Accession No. ATCC HB 58), and were linked tocyanogen bromide-activated Sepharose 4B for affinity purification ofproteins.

[0129] ACMNPV DNA and cationic liposomes (Invitrogen transfection kit#B825-03) or lipofectin (GIBCO/BRL #82925A) were used to transfect Sf9insect cells (American Type Culture Collection, Accession No. ATCC CRL1711; gift from Dr. Max Summers) in HyQ serum-free medium (Hyclone,#B-5502-L-P). BaculoGold (Pharmingen) was also used in sometransfections. Cells were maintained in TNM-FH medium (GIBCO BRL)supplemented with 10% fetal bovine serum and 0.1% F-68 pluronic acid(GIBCO/BRL, #670-404AG).

[0130] Construction of pVL941/mC_(k)

[0131] Based on the published sequence for the mouse kappa constantregion (mC_(k); Hieter, P. A., et al., Cell 22: 197-207 (1980)), twooligonucleotides having the following sequences (SEQ ID NO: 5 and SEQ IDNO: 6, respectively) were designed and synthesized: 5′ primer:    5′-GGATCC GCT GAT GCT GCA CCA ACT GTA TTC-3′ 3′ primer:     5′-CCTTTG TCC TAA CAC TCA TTC CTG TT-3′

[0132] The mC_(k) coding sequence was amplified by polymerase chainreaction (PCR) from the plasmid pVL91A3 containing the full-length 91A3mouse kappa light chain (Meek, K., et al., Proc. Natl. Acad. Sci.U.S.A., 84: 6244-6248 (1987)) An in-frame BamHI restriction site(underlined in the sequence above) was incorporated at the 5′-end of thecoding sequence to facilitate fusion with MAdCAM-1 sequences.

[0133] Vector pVL941 (Invitrogen) was linearized by digestion at sheunique BamHI site in the polyhedrin gene, and the ends were made bluntwith the Klenow fraqment of DNA polymerase. The amplified DNA fragment(312 base pairs) containing the 91A3 mouse kappa light chain was thenligated to pVL941 to yield pVL941/mC_(k). Clones containing the mC_(k)sequence in the same 5′ to 3′ orientation as the polyhedrin promoterwere selected.

[0134] Construction of Baculovirus Shuttle Vector

[0135] Based on the published nucleotlde sequence of murine MAdCAM-1(Briskin, M. J. et al., Nature, 363: 461-464 (1993)), oligonucleotideprimers having the following sequences were designed and synthesized:5′ primer:     5′-GAT CAG GGA TCC ATG GAA TCC ATC CTG GCC CTC CTG-3′3′ primer:     5′-TCG ATC GGA TCC GGT GGA GGA GGA ATT CGG GGT CA-3′

[0136] The ATG start codon of MAdCAM-1 is indicated in bold. The 5′ and3′ primers each incorporated a PamHI restriction site for cloning intothe expression vector (underlined above). Murine MAdCAM-1 sequences wereamplified by PCR using these 5′- and 3′-primers. The product wasdigested with BamEI and inserted into vector pVL941/mCk, which had beendigested with BamHI. DNA fragments cloned into the BamHI site werescreened for the correct orientation with respect to the mCk sequence.Fusion proteins produced from the resulting construct contained mouseMAdCAM-1 sequences at the N-terminus, and the mC_(k) sequence at thecarboxy-terminus (FIG. 3). A two-amino acid glycine-serine spacerencoded by the six nucleotides corresponding to the BamHI restrictionsite forms the junction between these sequences.

[0137] Transfection of Sf9 Insect Cells

[0138] Co-transfection (lipofection) of Sf9 insect cells with a mixtureof 1 μg of ACMNPV DNA (Invitrogen), 3-5 μg of shuttle vector DNA(pVL941/MAdCAM-1/mCk purified by magic-mini prep, Promega) and cationicliposomes (30 μl) was performed according to the manufacturers'instructions, with slight modifications. 2×10⁶ μ cells in TNM-FH medium(10% FBS) were plated out in 60 mm dishes and allowed to attach over a2-4 hour period prior to transfection. The medium was removed and theadherent cells were washed twice with serum-free medium (HyQ). Three mlof HyQ containing the DNA/liposome mixture were applied dropwise overthe cells; the cells were incubated overnight. The medium was thenremoved by aspiration and 5 ml TNF-FN medium containing 106 FBS wasadded to each dish. After 48 hours, one ml was removed for plaquepurification of recombinant virus, as previously described (O'Reilly, D.R., et al., (Eds.) Baculovirus Expression Vectors (W. H. Freeman andCo.: New York), pp. 124-128 (1992)).

[0139] ELISA Assav of Transfected Sf9 Cells For each transfLection,triplicate wells were coated with polyclonal goat anti-mC_(k) antibodies(2 μg/ml in carbonate/bicarbonate buffer), and incubated overnight. Theplates were washed three times with phosphate buffered saline (PBS)containing 0.5% Triton X-100, and 0.5 M NaCl and then washed twice withPBS. This washing protocol was used between all steps. The wells werethen blocked with 2% BSA in TBS for one hour. Supernatant (100 μl) takenfrom cells four to five days post-infection was aoplied to each well andincubated at 37° C. for 2 hours. The presence of mC_(k) fusion proteinwas detected by the addition of horseradish peroxidase-linked polyclonalanti-mC_(k) antibody and chromogenic substrate, according to standardprotocols.

[0140] Immunodetection of mC_(k) fusion proteins Recombinant viruscontaining the MAdCAM-1/mCk fusion gene was isolated by plaquepurification, and amplified by infecting Sf9 insect cells (2×10⁶cells/60 mm petri dish) in 5 ml TNM-FH medium. From this 5 ml stock, analiquot (100 μl) was taken to infect 2×10⁶ Sf9 cells as before.Supernatants (10 μl) taken 24, 48, 72 and 96 hours post-infection wereassayed for the presence, accumulation and stability of mC, fusionprotein by Western blot analysis. Samples were applied to 10% SDS/PAGEgels. Proteins were electrophoretically transferred to nitrocellulose bystandard techniques. The fusion protein was detected by addition ofpolyclonal goat anti-mC_(k)antibodies (2 μg) in 5 ml BLOTTO (5%)followed by treatment with alkaline phosphatase-linked swine anti-goat H& L chains and chromogenic substrate (BIORAD, Catalog No. 170-6432).

[0141] Purification of mC_(k) Fusion Protein by Affinity Chromatography

[0142] Production of fusion proteins was carried out in stirredmicrocarrer flasks. Sf9 insect cells (2×10⁶ cells/ml) were infected withvirus particles containing the MAdCAM-1/mCkfusion gene at a multiplicityof infection of 0.001. Upon complete lysis of the cells (approximatelyeight days later), the spent medium was clarified by low steedcentrifugation. The supernatant was applied to a rat monoclonalantli-mCk-coupled Sepharose column (purchased from Zymed or preparedusina rat monoclonal 187.1) equilibrated in TBS pH 7.0. The column waswashed with TBS pH 7.0, and the bound mC_(k) fusion protein eluted inglycine buffer pH 2.2 and neutralized by the addition of 2M Tris pH 8.0.Yields of 2 mg of mC_(k) fusion protein per liter of cells wereobtained.

[0143] Results

[0144] MAdCAM-1/mC_(k) fusion protein was expressed under thetranscriptional regulation of the viral polyhedrin promoter. To transferthe chimeric gene into the baculovirus genome, pVL941/MAdCAM-1/mC_(k)plasmid was co-transfected with A. californica nuclear polyhedrosisvirus DNA into Sf9 insect cells. Several recombinant plaques wereisolated and one was chosen for more detailed characterization.

[0145] ELISA assays on Transfected Cell Supernatants

[0146] To determine if an ELISA assay could be used to detect theappearance of fusion protein in the supernatant of transfected cells,aliquots (500 μl) were taken from the supernatant of transfected cellsat 3, 4 and 5 days post-transfection, and ELISA assays were performed asdescribed above. The results indicated that the sensitivity of the ELISAassay was sufficient to detect the mC_(k) fusion protein. The assay canbe used to provide an indication that the desired gene is expressed inthe insect cells. Moreover, a positive signal in the ELISA assaycorrelated well with the isolation of recombinant viral particles insubsequent plaque assays.

[0147] Cellular Adhesion Assay

[0148] The Following buffers and reagents were used in the initialtitration of capture antibody and in the adhesion inhibition assays:TABLE 1 CARBONATE BUFFER: 17.2 g NaHCO₃ Sigma #S-5575 8.6 g Na₂CO₃ Sigma#S-6139 Bring volume to 1L with H20 1% BSA/PBS BSA 5 g Sigma #A-6793 PBS500 ml GIBCO #14040-026 Sterile filter ASSAY BUFFER (HBSS/2% FCS/ 25mMHEPES/ Pen/Strep., pH 7.2): HBSS 500 ml Gibco #14025-02 FCS 10 ml Gibco#16000-044 HEPES (1M) 12.5 ml Gibco #15630-015 Pen/Strep. (100X) 5 mlGibco #15070-022 Sterile filter 2X ASSAY BUFFER (2X HBSS/4%FCS/ 50mMHEPES/ Pen/Strep., pH 7.2): 10x HBSS 100 ml Gibco #14065-023 FCS 20 mlGibco #16000-044 HEPES (1M) 25 ml Gibco #15630-015 NaHCO₃(7.5%) 4.7 mlGibco #25080 Pen/Strep. 10 ml Gibco #15070-022 Check pH Bring volume upto 500 ml with H2O Sterile filter BCECF-AM Molecular #3-1170 Probes DMSOSigma #D-8779

[0149] Maintenance and Labelling of Cells

[0150] HUT 78 cells (a human T cell lymphoma line; American Type CultureCollection, Accession No. ATCC TIB 161) were maintained in culture forup to one month in RPMI 1640 supplemented with 10% FCS, 20 mM HEPES, andPen-Strep (1:100). Just prior to use in adhesion assays, cells werelabeled with BCECF-AM as follows: 1×10⁷ cells were pelleted at 1000 rpmfor 10 minutes and resuspended in 25 mL cold PBS (Phosphate BufferedSaline). The cells were pelleted again, resuspended in cold PBS, andpelleted again. The cell pellet was resuspended in 25 ml PBS and labeledwith 50 μl BCECF-AM (1 μg/μL in DMSO) for 30 minutes at 37° C.(BCECF-AM; 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyflourescein,acetoxymethyl ester). The labeled cells were pelleted at 700 rpm for 10minutes, resuspended in 25 ml cold Assay Buffer and pelleted. Finally,labeled cells were counted and resuspended in Assay Buffer (ambienttemperature) at a concentration of 2.5×10⁶/ml.

[0151] Titration of Capture Antibody

[0152] A rat anti-mCk antibody (affinity purified rat-anti-mC_(k)monoclonal antibodies from rat hybridoma cell line 187.1; ATCC AccessionNo. HB58) was selected for use as capture antibody in the adhesionassay. A titration experiment was conducted in order to determine theoptimal capture antibody concentration. Assay Buffer for the titrationwas 2X Assay Buffer.

[0153] ELISA plates were coated with various concentrations captureantibody by adding 10 μg/ml, 5 μg/ml, 2.5 μg/ml, or 1.25 μg/ml ofantibody in a 50 μl volume to each well and incubating at 4° C.overnight. The next day, plates were blocked with 100 μl 1% BSA/PBS at37° C. for 1 hour. After one hour, the BSA/PBS solution was removed, and50 μl MAdCAM-mCk fusion protein (neat supernatant) was added to eachwell and incubated for 1 hour at 37° C.

[0154] Anti-murine MAdCAM-1 antibody MECA-367 (American Type CultureCollection, Accession No. ATCC HB 9478) was used as a blocking antibody.For the titration, either 20 μl of Assay Buffer (i.e., 2X Assay Buffer),20 μl of neat supernatant containing anti-MAdCAM-1 MECA-367 antibody, or20 μl of neat supernatant containing MECA-367 diluted 1:2, 1:4, 1:8 or1:16 in Assay Buffer, was added to each well. The undiluted supernatantcontained approximately 3 μg/ml of antibody.

[0155] Frozen BCECF-labeled HUT 78 cells were thawed and resuspended inAssay Buffer at 2.5×10⁶ cells/ml. 50 μl of cells, 50 μl of Assay Buffer(without Pen/Strep), 30 μl of water, 50 μl of 8 mM MnCl₂, and 20 μl ofeither MECA-367 antibody (neat or diluted) or 20 μl of Assay Bufferalone, were then added to each well, and the plates were incubated on arotator for 30 minutes at room temperature.

[0156] After incubation, a baseline measurement for each well of totalfluorescence was taken using a Fluorescence Concentration Analyzer(IDEXX) at 485/535 nM. Then plates were washed twice with a solution of50 mM Tris/2 mM MnCl. using an EL 404 Microplate Autowasher (BIO-TEKInstruments) , and fluorescence (due to adherent cells) was determinedagain using a Fluorescence Concentration Analyzer (IDEXX) at 485/535 nM.For each well, the final reading was divided by the baseline reading.The values from duplicate wells were averaged, and plotted (FIG. 1).Accordingly, 50 μl of a 5 μg/mL solution of rat anti-mCk was used tocoat each well for subsequent adhesion assays.

[0157] Inhibition Assays and IC₅₀ Determination

[0158] The assay was performed in a 96-well format. Plates were coatedovernight at 4° C. with 53 μl/well of a 5 μg/mL solution of rat anti-mCkin carbonate buffer. The plates were blocked for 1 hour at 37° C. with100 μL/well of 1% BSA in PBS. The BSA/PBS solution was removed, and 50μL of MAdCAM-mCk supernatant was added neat to each well. The assay wasconducted under conditions in which capture antibody was the limitingreagent.

[0159] Cell adhesion was measured in the presence or absence ofcompounds. The compounds were resuspended in 100% DMSO, subsequentlydiluted 1:10 in water, and finally diluted again 1:10 in the assay: 50μL of 2X Assay Bufler, 30 μL water, 20 μL of compound were added to eachwell. 50 μL of a cell suspension containing BCECF-AM labeled HUT 78cells (resuspended in assay buffer at a concentration of 2.5×10⁶/ml; seeabove) were added to each well, followed by 50 μL of a solution of 8 mMMnCl₂ in assay buffer. Cell adhesion occurred in 30 minutes at ambienttemperature, after which plates were washed with 50 mM Tris/2 mM MnCl₂,pH 7.2 using an EL 404 Microplate Autowasher (BIO-TEK Instruments) usingthe following wash parameters: wash volume=500 μL; two wash cycles; washdepth=80; aspirating after each wash. Plates were read using aFluorescence Concentration Analyzer (IDEXX) at 485/535 nM.

[0160] IC₅₀ Determinations

[0161] In order to determine an IC₅₀ (the concentration of compoundwhich inhibits 50% of the total number of cells adhering to MAdCAM-1relative to a control conducted in the absence of inhibitor), compoundswere tested for inhibition of HUT 78 cell adhesion as described above atvarious concentrations ranging between 62.5 to 500 μM. Additionaldeterminations were conducted as needed, so that each IC₅₀ wasdetermined over a range which encompassed the IC₅₀. Over this range,Four different concentrations were tested in duplicate. For example,compound 793-1a was tested at four different concentrations (50 μM, 25μM, 12.5 μM and 6.25 μM) in duplicate wells.

[0162] For each compound, a ratio was determined as follows: An averageof the readings from duplicate wells was divided by an average of eightcontrol wells (adhesion in the absence of any compound). Percentinhibition of adhesion was calculated as 100×(1−the ratio). In addition,the concentration (μM) of compound used was plotted against theresulting ratios. The resulting plot for compound 793-1a is shown inFIG. 2.

[0163] The IC₅₀ was then determined using Kaleidograph software(Abelbeck Software). The complete procedure was repeated again usingduplicate wells for each point. An average of two IC₅₀ determinationswas obtained, and the resulting values are presented in Table 2 withpercent adhesion inhibition for a variety of compounds of the presentinvention. For example, the IC₅₀ for compound 793-1a was determined tobe 14 μM. Cysteines in cyclic peptides which are linked by a disulfidebond are indicated by an “*”.Abbreviations used are defined in Table 3below.

[0164] In Table 2, Example No. 1 is represented by SEQ ID NO: 7; ExampleNo. 2 is represented by SEQ ID NO: 8; Example No. 3 is represented bySEQ ID NO: 9; Example No. 4 is represented by SEQ ID NC: 10; Example No.5 is represented by SEQ ID NO: 11; Example No. 6 is represented by SEQID NO: 12; Example No. 7 is represented by SEQ ID NO: 13; Example No. 8is represented by SEQ ID NO: l4; Example No. 9 is represented by SEQ IDNO: 15; Example No. 10 is represented by SEQ ID NO: 7; Example No. 11 isrepresented by SEQ ID NO: 16; Example No. 12 is represented by SEQ IDNO: 17; Example No. 13 is represented by SEQ ID NO: 18; Example No. 14is represented by SEQ ID NO: 9; Example No. 15 is represented by SEQ IDNO: 13; Example No. 16 is represented by SEQ ID NO: 19; Example No. 17is represented by SEQ ID NO: 20; Example No. 18 is represented by SEQ IDNO: 21; Example No. 19 is represented by SEQ ID NO: 22; Example No. 20is represented by SEQ ID NO: 23; Example No. 21 is represented by SEQ IDNO: 24; Example No. 22 is represented by SEQ ID NO: 25; Example No. 23is represented by SEQ ID NO: 26; Example No. 24 is represented by SEQ IDNO: 7; Example No. 25 is represented by SEQ ID NO: 27; Example No. 26 isrepresented by SEQ ID NO: 28; Example No. 27 is represented by SEQ IDNO: 29; Example No. 28 is represented by SEQ ID NO: 30; Example No. 29is represented by SEQ ID NO: 31; Example No. 30 is represented by SEQ IDNO: 32; Example No. 31 is represented by SEQ ID NO: 33; Example No. 32is represented by SEQ ID NO: 34; Example No. 33 is represented by SEQ IDNO: 35; Example No. 34 is represented by SEQ ID NO: 36; Example No. 35is represented by SEQ ID NO: 37; Example No. 36 is represented by SEQ IDNO: 38; Example No. 37 is represented by SEQ ID NO: 39;

[0165] Example No. 38 is represented by SEQ ID NO: 40; Example No. 39isple No. 40 is represented by SEQ ID NO: 42; Example No. 41 is represented by SEQ ID NO: 43; Exampl e No. 42 is represented by SEQ IDNO: 44; Example No. 43 is represented by SEQ ID NO: 45; Example No. 44is represented by SEQ ID NO: 46; Example No. 45 is rep resented by SEQ ID NO: 47; Example No. 46 is represented by SEQ ID NO: 48; Example No. 47is represented by SEQ ID NO: 49; Example No. 48 is represented by SEQ IDNO: 50; Example No. 49 is represented by SEQ ID NO: 51; Example No. 50is represented by SEQ ID NO: 52; Example No. 51 is represented by SEQ IDNO: 53; Example No. 54 is represented by SEQ ID NO: 54; Example No. 55is represented by SEQ ID NO: 55; Example No. 56 is represented by SEQ IDNO: 56; Example No. 57 is represented by SEQ ID NO: 57; Example No. 58is represented by SEQ ID NO: 7; Example No. 59 is represented by SEQ IDNO: 58; Example No. 60 is represented by SEQ ID NO: 59; Example No. 61is represented by SEQ ID NO: 60; Example No. 62 is represented by SEQ IDNO: 61. TABLE 2 % % Adhesion Method MH + Purity Inhibition IC50 Ex No L#Sequence Prep Cal. Found HPLC 500 μM (μM) 1 00112-1AAc-Leu-Asp-Thr-Ser-Leu B 591 616 75 278 2 00619-1AAc-Leu-Asp-Ala-Ser-Leu B 557 563 78 47 1004 3 0092-1AAc-His-Trp-Arg-Gly-Leu-Asp-Thr B 926 926 50 49 584 4 00167-1BAc-Cys*-Leu-Asp-Thr-Ser-Leu-Cys* B,D 797 816 67 51 (@250 μM) 102 .4 500145-1B Ac-Cys*-Gly-Leu-Asp-Thr-Ser-Leu-Gly-Cys* B,D 909 925 80 45(@200 μM) 266 6 0093-1A Ac-Trp-Arg-Gly-Leu-Asp-Thr-Ser B 877 878 42 44862 7 0096-1A Ac-Trp-Arg-Gly-Leu-Asp-Thr B 789 792 53 46 725 8 0094-1AAc-Arg-Gly-Leu-Asp-Thr-Ser-Leu B 804 802 34,41 35 1575 9 0099-1AAc-Gly-Leu-Asp-Thr-Ser-Leu B 648 647 67 58 339 10 0104-1AAc-Leu-Asp-Thr-Ser-Leu B 591 616 47 50 530 11 0093-1AAc-Trp-Arg-Gly-Leu-Asp-Thr-Ser B 877 878 42 44 862 12 0097-1AAc-Arg-Gly-Leu-Asp-Thr-Ser B 691 691 32,34 31 1789 13 0108-1AAc-Gly-Leu-Asp-Thr-Ser B 535 554 73 43 745 14 0092-1AAc-His-Trp-Arg-Gly-Leu-Asp-Thr B 926 926 50 49 584 15 0096-1AAc-Trp-Arg-Gly-Leu-Asp-Thr B 789 792 53 46 725 16 0105-1AAc-Arg-Gly-Leu-Asp-Thr B 603 607 45 48 542 17 00379-1AAc-MeLeu-Leu-Asp-Thr-Ser-Leu B 965 966 50 28 1316 18 00380-1APhx-Leu-Asp-Thr-Ser-Leu B,C 722 722 32 818 19 00381-1AImpa-Leu-Asp-Thr-Ser-Leu B,C 736 737 44 1140 20 00382-1ABipa-Leu-Asp-Thr-Ser-Leu B,C 742 765 80 54 485 21 00383-1ANpa-Leu-Asp-Thr-Ser-Leu B,C 716 713 38,48 39 548 22 00384-1AHbz-Leu-Asp-Thr-Ser-Leu B,C 750 770 77 51 401 23 00386-1APba-Leu-Asp-Thr-Ser-Leu B,C 818 817 94 88 (@50 μM) 2.8 24 00389-1AAc-Leu-Asp-Thr-Ser-Leu B,C 710 711 61 29 205 25 00391-1ATpc-Leu-Asp-Thr-Ser-Leu B,C 672 670 88 39 121 26 00392-1APha-Leu-Asp-Thr-Ser-Leu B,C 666 693 86 38 2128 27 00393-1AMeLeu-Leu-Asp-Thr-Ser-Leu B,C 905 928 86 54 256 28 00710-1ATpa-Leu-Asp-Thr-Ser-Leu B,C 813 837 74 0.3 29 00711-1APaa-Leu-Asp-Thr-Ser-Leu B,C 785 790 81 0.5 30 00781-1APba-Pro-Leu-Asp-Thr-Ser-Leu B,C 910 912 77 47 31 00784-1ADpa-Leu-Asp-Thr-Ser-Leu B,C 737 765 87 11 32 00785-1ADphb-Leu-Asp-Thr-Ser-Leu B,C 799 807 87 15 33 00786-1AFlc-Leu-Asp-Thr-Ser-Leu B,C 735 739 71 17 34 00787-1APca-Leu-Asp-Thr-Ser-Leu B,C 771 799 80 2.1 35 00788-1AXnc-Leu-Asp-Thr-Ser-Leu B,C 751 757 84 3 36 00789-1APba-Leu-Asp-Thr-Ser-Leu B,C 813 818 81 4 37 00793-1ATphp-Leu-Asp-Thr-Ser-Leu B,C 823 831 87 14 38 00795-1A2-Anc-Leu-Asp-Thr-Ser-Leu B,C 747 756 63 5.4 39 00603-1APba-Leu-Ala-Thr-Ser-Leu B,C 771 775 84 100 (@250 μM) 28 40 00605-1AAc-Leu-Phe-Thr-Ser-Leu B 623 623 86 29 299.7 41 00606-1AAc-Leu-Glu-Thr-Ser-Leu B 605 605 85 98 60.7 42 00610-1APba-Leu-Tyr-Thr-Ser-Leu B,C 862 867 95 100 30 43 00612-1AAc-Leu-Asp-Ser-Ser-Leu B 578 580 86 38 611.4 44 00613-1AAc-Leu-Asp-Thr-Thr-Leu B 604 609 71 98 100 .7 45 00614-1AAc-Leu-Asp-Thr-Ser-Phe B 625 625 80 86 143.9 46 00615-1AAc-Lle-Asp-Thr-Ser-Leu B 591 593 79 17 18983 47 00712-1AAc-Leu-Asp-Val-Ser-Leu B 589 611 93 71 48 00716-1AAc-Leu-Asp-HyP-Ser-Leu B 599 626 79 106 49 00718-1AAc-MeLeu-Asp-Thr-Ser-Leu B 587 629 65 150 50 00799-1ACpc-Asp-Thr-Ser-Leu B,C 526 532 73 8.9 51 00649-1A Pba-Asp-Thr-Ser-LeuB,C 702 704 85 100 (@100 μM) 13.3 52 00800-1A Pca-Asp-Thr B,C 459 460 7611 53 00650-1A Pba-Asp-Thr B,C 503 505 96 100 (@100 μM) 13.5 54NH2-Leu-Asp-Thr-Ser-Leu 22 55 NH2-Trp-Arg-Gly-Leu-Asp-Thr-Ser-Leu- 76Gly-Ser 56 NH2-His-Trp-Arg-Gly-Leu-Asp-Thr-Ser- 80 Leu-Gly-Ser-Val 57NH2-Arg-Val-His-Trp-Arg-Gly-Leu-Asp- 90 Thr-Ser-Leu-Gly-Ser-Val-Gln 58Ac-Leu-Asp-Thr-Ser-Leu 71 59 Ac-Arg-Gly-Leu-Asp-Thr-Ser-Leu-Gly 75 60Ac-Trp-Arg-Gly-Leu-Asp-Thr-Ser-Leu-Gly- 71 Ser 61Ac-His-Trp-Arg-Gly-Leu-Asp-Thr-Ser-Leu- 71 Gly-Ser-Val 62Ac-Arg-Val-His-Trp-Arg-Gly-Leu-Asp-Thr- 88 Ser-Leu-Gly-Ser-Val-Gln

[0166] TABLE 3 Ac = acetyl Anc = 2 Anthracenecarbonyl Bipa =4-biphenylacetyl Bpc = benzofuranecarbonyl Chc = cyclohexanecarbonyl Cpa= 1-cyclopentylacetyl Cpc = cyclopentanecarbonyl DBU = diazobicyclo[5,40] undec-7-ene DMF = N,N-dimethylformanide Dpa = diphenylacety Dphb= 3,5-diphenylbenzoyl EDT = 1,2-ethanedithiol Fc = furanecarbonyl HBTU =2(1-benzotriazolyl-1-yl-)-1,1,3,3-tetramethyluronium Hbz =4-heptylbenzoyl Hyp = 4-hydroxyproline Idc = Indolecarbonyl Impa =4-isobutyl-a-methylphenylacetyl Indc = Indanecarbonyl NMM =N-methylmorpholine Npa = a-naphthylacetyl Paa = 1-pyreneacetyl Pba =1-pyrenebutyryl Pca = 1-pyrenecarbonyl Pha = phenylacetyl Phx =6-phenylhexanoyl PYBOP = benzotriazolyl-N-oxytripyrrolidinophosphoniumhexafluorophosphate Rink Amide Am Resin =4-[2′,4′-dimethoxyphenyl-Fmoc-aminomethyl]-phenoxylacetamido-norlecucylaminomethyl resin Tcc =trans-cinnamoyl TFA = trifluoroacetic acid Thc = Thienylcarbonyl Tpa =triphenylacetyl Tpc = tetramethylcyclopropylcarbonyl Tphp =triphenylpropionyl Xnc = xanthenecarbonyl

Example 3: Human MAdCAM Adhesion Assay Design and Functional Analysis ofa Human MAdCAM-1-IgG (huMAdCAM-1-Ig) Chimera

[0167] Construction of huMAdCAM-IgG Chimera

[0168] Human MAdCAM-1 clone 4 cDNA (FIG. 4, SEQ ID NO: 67), present invector pCDNA3 (Invitrogen, San Diego, Calif.), was used as a templatefor PCR amplification of extracellular regions of human MAdCAM-1 to befused with the constant region of a human IgGl. The human MAdCAM-1 clone4 cDNA/pcDNA3 construct is also referred to as pcD3huMAd4 or pCDhuMAd4(see Briskin et al., WO 96/24673, published Aug. 15, 1996, Example 1;see also Shyjan, A. M. et al., J. Immunol., 156: 2851-2857 (1996), theteachings of which are each incorporated herein by reference). Inparticular, primer HUMADIG4/2 (SEQ ID NO: 83), which contains the 5′ endof human MAdCAM-1 coding sequence (ATO codon, bold), was synthesized:

[0169] HUMADIG4/2 (SEQ ID NO: 83),

[0170] HindIIl

[0171] 5′-GGAAGCTTCCACCATGGATTTCGCACTGGCCC-3′

[0172] This 5′ primer was used in conjunction with a 3′ primer toamplify a region encoding the entire extracellular domain of humanMAdCAM-1 (clone 4). The 3′ primer, designated HUMADIG3 (SEQ ID NO: 84),which contains a portion complementary to coding strand nucleotides992-1010 of SEQ ID NO: 67, had the following sequence:

[0173] HUMADIG3 (SEQ ID NO: 84)      SpeI5′-GGACTAGTGGTTTGGACGAGCCTGTTG-3′

[0174] The primers were designed with a 5′ HindIII site or 3′ SpeI sitesas indicated. These primers were used to PCR amplify a MAdCAM fragment,using a PCR optimizer kit from Invitrogen (San Diego, Calif.). The PCRproduct was digested with the enzymes HindIII and SpeI to generate endsfor cloning, and was purified by gel electrophoresis using the GlassmaxDNA isolation system (Gibco, Bethesda, Md.).

[0175] A −1 kb fragment encompassing the CH1, H (hinge), CH2 and CH3constant regions was excised by digestion with SpeI and EcoRI from aconstruct encoding a human immunoglobulin γ1 heavy chain, having aconstant region described by Reichmann, L. et al., Nature, 322: 323-327(1988) and Takahashi, N. et al., Cell, 29: 671-679 (1982)), whichfurther contained two mutations in the Fc region. The mutations in theFc region of this Ig heavy chain construct (Leu²³⁵→Ala²³⁵ andGly²³⁷→Ala²³⁷) were designed to reduce binding to human Fcγ receptors,and were produced by oligonucleotide-directed mutagenesis. The antibodyencoded by this construct was used as an isotype matched irrelevantcontrol hereinbelow. The 1 kb SpeI-EcoRI fragment encoding theFc-mutated IgG1 constant region was isolated by gel electrophoresisusing the Glassmax DNA isolation system (Gibco, Bethesda Md.). Thisconstant region fragment and the HindIII-SpeI fragment containing theentire extracellular domain were ligated in a three-way ligation tovector pEE12 (Stephens, P. L. and M. L. Cockett, Nucl. Acids Res., 17:7110 (1989) and Bebbington, C. R. and C. C. G. Hentschel, 1987, The useof vectors based on gene amplification for the expression of clonedgenes in mammalian cells, (Academic Press, N.Y.)), which had beendigested with HindIII and EcoRI. Transformants of the bacterial strainDH10B were obtained. Colonies were grown and mini plasmid preps wereanalyzed by restriction mapping. A construct designated HuMAdIg21 (fromclone 21), which encodes a fusion protein comprising the entireextracellular domain of MAdCAM-1 fused to the Fc-mutated IgG1 constantregion, was sequenced across the entire MAdCAM-1 portion, confirmingproper fusion of segments and the absence of PCR induced mutations.

[0176] For initial testing, the construct was transiently transfectedonto monolayers of 5×10⁷ COS cells in 1 ml of RPMI buffer (no serum) and25 μg of plasmid using electroporation with a Biorad Gene Pulser understandard conditions (960 μF, 250 V). 72-96 hours after transfection,supernatants were harvested, passaged through 0. 45 μ filters and storedat 40° C. in the presence of 0.05% sodium azide. Production of chimerawas confirmed by a sandwich ELISA, using an anti-human IgG1 antibody ascapture antibody and the same antibody, which was conjugated to alkalinephosphatase as second antibody for detection. Irrelevant controlantibody (having an identical constant region) was used as a standard.The chimera was also analyzed by Western blotting using an anti-humanMAdCAM-1 monoclonal antibody, and was found to run at approximately 200kd, consistent with the size of a homodimer.

[0177] Soluble Human MAdCAM-Ig Chimeras Specifically Bind α4β7 PositiveCells

[0178] Supernatants from two different transfections were assayed fortheir ability to stain the T cell line HUT 78, which was previouslyshown to bind MAdCAM-1 only in the presence of Mn++. Accordingly, eachsolution used in this assay contained 2 mM Mn++. HUT 78 cells (a human Tcell lymphoma line; American Type Culture Collection, Accession No. ATCCTIB 161) are α4β7-bearing cells. To test the binding specificity of thechimeras, HUT 78 cells were preincubated with either media alone (RPMI1640 with 2% FCS) or media and 10 μg/ml of the anti-β7 antibody FIB 504.Approximately 100,000 cells were incubated on ice for 15 minutes andthen washed with HBSS plus 2% FCS/2 mM Ca++/2 mM Mn++. Cells were thenincubated for 20 minutes on ice with media once again, or withsupernatants from one of two independent transfections with theconstruct encoding the chimera comprising the entire extracellulardomain of MAdCAM-1 (HuMAdIg21). After washing, cells were then incubatedwith an anti-human IgG antibody conjugated with phycoerythrin andstaining above background was assessed by flow cytometry (FACScan). Onlycells incubated with the chimera supernatants stained above background,while preincubation with the β7 MAb reduced this staining to backgroundlevels, indicating a specific interaction of the chimera with the α4β7integrin.

[0179] Permanent NSO cell lines secreting human MAdCAM-Ig chimera wereselected after transfection by electroporation, by growth in a glutaminefree media as previously described (Cockett, M. L., et al.,Bio/Technology, 8: 662-667 (1990)). Cloned lines were adapted to growthin spinner culture. Supernatants from three of these cloned lines(samples B-D), and a partially purified chimera (Clone 21, purified bybinding to protein A, sample A) were tested for their ability to supportadhesion of the B cell line RPMI 8866. Briefly, NEN maxisorb plates wereincubated with 100 μL/well of Protein A at 20 μg/ml in carbonate buffer,pH 9.5 overnight at 4° C. Plates were then washed 2X with RPMI 1640media (no serum). 100 μl of chimera (or serial dilutions in RPMI) werebound to the wells at 37° C. for 2 hours and then washed once. Wellswere then blocked with FCS for 1 hour at 37° C., washed once, and thenpreincubated with tissue culture supernatants containing either ananti-human VCAM-1 MAbs (2G7) as a control or the anti-human MAdCAM-1 MAb10G3 (Briskin et al., WO 96/24673, Example 2). 2G7 and 10G3 MAbs wereremoved before addition of cells. RPMI 8866 cells were fluorescentlylabeled by preincubation with BCECF-AM stain (BCECF-AM;2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyflourescein, acetoxymethylester; Molecular Probes), 100 μl of cells were added to each well (to afinal concentration of 10⁵ cell/well), and incubated on a rotary shakerfor 30 minutes at room temperature. Binding of RPMI 8866 cells toimmobilized chimeras was assessed by reading flourescence values using aFluorescence Concentration Analyzer (IDEXX). Specific binding wasdemonstrated as only the anti-human MAdCAM-1 MAb could block binding ofcells to MAdCAM-Ig chimera (Table 3A). TABLE 3A Anti HuMAd Sample MAbsNeat 1:2 1:4 1:8 1:16 1:32 A − 195527 195527 195527 18560 4254 3558 +3860 3092 1746 2200 482 564 B − 195527 195527 195527 195528 52056 2932 +6526 3626 3274 2978 1648 1518 C − 195527 195527 35548 9570 21782 1926 +4566 4094 3922 3492 2436 2566 D − 195527 30840 46852 16270 5474 2656 +7350 6794 6020 4510 6548 5122 #Purified chimera or supernatants (usedeither undiluted (“neat”) or at serial 1:2 dilutions) was bound to wellsvia Protein A, and incubated with fluorescently-labeled RPMI 8866 cellson a rotary shaker for 30 minutes at room temperature. After washingwith an automated plate washer (EL 404 Microplate Autowasher, BIO-TEKInstruments), bound cells were counted with an automated plate reader(IDEXX). Raw numbers are thus a reflection of numbers of cells bound.

[0180] Production and Purification of huMAdCAM-1-Ig Chimera

[0181] NS0 clone 21-9C10 producing huMAdCAM-1-Ig from HuMAdIG21 wasprepared as described above. The clone was grown in glutamine free DMEM,10% ultra low bovine Ig FCS, 1X GS supplement (Cibco Inc.), (a mixtureof non-essential amino acids and nucleosides for addition toglutamine-free basal media), 1 mM pyruvate, 50 units/ml penicillin G and50 μg/ml streptomycin sulfate. huMAdCAM-1-Ig producing clones wereadapted to grow in 1.0 liter spinner flasks at 37° C., 5% CO₂, 95s airand a constant stir rate of 60-70 rpm. Cultures were seeded at a celldensity of 1 to 2×10⁵ cells/ml in 250 mls of the above media and allowedto grow for 48-72 hours before bringing the final volume of the cultureto 550 to 600 mls. Spinner flasks were harvested at day 10-11 andcentrifuged at 6,000 rpm, 4° C., 25 minutes. The resulting culturesupernatants were then 0.2 μ filtered and stored at 4° C.

[0182] huMAdCAM-1-Ig culture supernatants were passed over a 20 ml bedvolume Protein A column, at equilbrated in PBS, pH 7.2, at 1.5 mls/minflow rate. The column was then washed at 5.0 mls/min with PBS, pH 7.2 at1.5 mls/min flow rate. The column was then washed at 5.0 mls/min withPBS pH 7.2 and eluted with 0.1 M citrate, pH 3.5 at a flow rate of 5.0mls/min. 5.0 ml fractions were collected and neutralized immediatelywith 250 μl/fraction 1.5 M sodium carbonate, pH 11.5. The Protein Acolumn was then equilibrated with PBS, pH 7.2.

[0183] The resulting fractions from Protein A purification were analyzedby an anti-human Ig(Fc) (Jackson Research Labs.) ELISA. Peak fractionscontaining huMAdCAM-1-Ig were concentrated by centrifugacion at 6,000rpm, 4° C. with Centricon 30 concentrators (30,000 mw cutoff).

[0184] Final product was quantitated by BioRad Protein Assay, analyzedfor biological activity in the huMAdCAM-1-Ig/RPMI 8866 Adhesion Assay(see below) and for purity with 4-20% SDS-PAGE, reduced and nonreduced,by colloidal coomassie blue staining.

[0185] Adhesion Assay Protocol

[0186] RPMI 8866 cells, a B cell lymphoma expressing α4β7 (a gift fromD. Erle; Erle, D. J. et al., J. Immunol., 153:517-528 (1994); Shyjan etal., Journal of Immunology, 1996:2851 (1996) were fluorescently labeled(labeling protocol provided below) using BCECF (Molecular probes#B-1170). Cells were resuspended in an assay buffer consisting of HanksBalanced Salt Solution (HBSS) supplemented with fetal calf serum (FCS)at a final concentration of 2%, HEPES, pH 7.3 at 25 mM. All reagents inthis buffer were supplied from Gibco/BRL.

[0187] All assays were performed in 96 well strip well plates fromCostar (E.I.A./R.I.A. Strip plate-8 Cat. #2581). Protein A purifiedhuman MAdCAM-1-Ig chimera was suspended in carbonate buffer (0.2 MNaHCO₃ and 0.8 M Na₂CO₃, pH 9.5) at a concentration of 200 ng/ml and 50μl was added to each well for a plating concentration of 10 ng/well. Theplates were incubated at 37° C. for one hour (or at 4° C. overnight) andwashed once in the automatic plate washer (50 mM Tris HCl, 0.14 M NaCland 2.0 mM MnCl₂ at pH 7.2) (Bio-Tek EL 404 microplate autowasher).Wells were then blocked with PBS (phosphate buffered saline) and 10% FCS(100 μl/well) for one hour at 37° C. followed by the same wash step asdescribed.

[0188] Cells were labeled with BCECF-AM (Molecular Probes, Cat. No.B-1170) at a concentration of 2 μl/ml to a cell suspension of 4×10⁶cells/ml in PBS. The cells were incubated in a 37° C. water bath for 30minutes. Labeled cells were centrifuged at 700 RPM for 10 minutes,resuspended in assay buffer and the centrifugation step was repeated.The cells were resuspended at a final concentration of 2.5×10⁶ cells/ml.

[0189] For assays, compounds were diluted as follows in a 96 wellpolypropylene plate. First, a stock solution of each compound wasprepared by dissolving compound in 100% DMSO at 100X final concentrationto be tested. 10 μl of the stock was added to 90 μl water for the firsttest dilution. 50 μl of the first test dilution was added to 50 μl of10% DMSO in water for the second test dilution. 50 μl of the second testdilution was added to 50 μl of 10% DMSO in water for the third testdilution. 50 μof the third test dilution was added to 50 μl of 10% DMSOin water for the fourth test dilution, and so on, until six testdilutions were prepared.

[0190] A stock solution of SmM positive control peptide (L783-1A) wasmade in 100% DMSO, and subsequently diluted in water 1:10 (10 μl of 5 mMstock plus 90 μl of water) to yield a concentration of 0.5mM. Thisdilution was then further diluted 1:2 with 10% DMSO (50 μl of thecompound and 50 μl of 10% DMSO) for a concentration of 250 μM. These 1:2dilutions were continued serially until six dilutions were achieved.These dilutions represent 10X stocks as shown in the assay set up below:

[0191] The assay was set up by adding materials to huMAdCAM-1-Ig-coatedplates as follows:

[0192] 1) 130 μl/well of assay buffer

[0193] 2) 20 μl/well of diluted compounds or 10% DMSO (control) ordiluted positive control peptide.

[0194] 3) 50 μl of ECECF-AM-labeled RPMI 8866 cells

[0195] In this example, the final concentration of cells was 1.25×10⁶cells/well and the final concentration of compound ranged in a six pointassay from 50 μm to 1.06 μm.

[0196] The plates were wrapped in foil and incubated for 30 minutes atroom temperature on a rotary shaker at 45 RPM.

[0197] Plates were washed twice (50 mM Tris HCl, 0.14 mM NaCl and 2.0 mMMnCl₂ at pH 7.2) on the microplate autowasher plate washer set asfollows:

[0198] Wash Volume=500 ml

[0199] Wash Cycle=2X

[0200] Soak Time=0

[0201] Wash Depth=80

[0202] Aspirate After Last Wash

[0203] Shake Time=0

[0204] Plates were read in an IDEX fluoresecent plate reader set toexcite at 485 nm and read at 535 nm.

[0205] Data was collected in microsoft excel in a format for automaticIC 50 determinations. The IC 50 is the fluorescence level that is 50% ofmaximum binding. Results are shown in Tables 4-7.

[0206] In Table 4, Example No. 1478 is represented by SEQ ID NO: 71;Example No. 783 is represented by SEQ ID NO: 72; Example No. 1492 isrepresented by SEQ ID NO: 73; Example No. 1487 is represented by SEQ IDNO: 74; Example No. 1490 is represented by SEQ ID NO: 75; Example No.1275 is re prese nted by SEQ ID NO: 76; Example No. 1274 is representedby SEQ ID NO: 77; Example No. 1282 is represented by SEQ ID NO: 78;Example No. 1481 is represented by SEQ ID NO: 79; Example No. 1482 isrepresented by SEQ ID NO: 80; Example No. 1486 is represented by SEQ IDNO: 81; Example No. 1498 is represented by SEQ ID NO: 82. TABLE 4R₁-Leu-Asp-Thr-Ser-Leu Method Mass Mass hMAdCAM L # R₁ Prep Calc. FoundIC50 1478

C 647 658 11.2  783

C 673 701 10 1492

C 687 715 2.1 1487

C 686 690 3 1490

C 720 724 1.7 1275

C 698 726 3.5 1276

C 698 703 2.7 1282

C 699 706 4 1481

C 698 705 3.2 1482

C 698 700 2.2 1486

C 698 705 1.4 1491

C 649 658 3.4

[0207] TABLE 5 R₁-Leu-Asp-Thr Method Mass Mass hMAdCAM L # R₁ Prep Calc.Found IC50 1496

C 448 503 8.2 1495

C 499 501 3.1 2277

C 584 587 1.7 2278

C 570 575 0.4 2273

C 618 620 1.2 2271

C 607 601 0.9 2261

C 632 633 0.86 2263

C 621 622 1.3 2262

C 654 679 1.5 2279

C 632 636 1

[0208] TABLE 6 R₁-Leu-Asp-Thr Method Mass Mass hMAdCAM Example R₁ PrepCalc. Found IC50 1

C 550 550 0.58 2

C 518 518 0.73 3

C 484 484 1.4 4

C 506 506 1.1 5

C 493 493 1.5 6

C 535 534 2.6 7

C 518 518 2.8 8

C 486 486 2 9

C 499 499 1.1

[0209] TABLE 7 R₁-Leu-Asp-Thr-R₂ Method Mass Mass hMAdCAM ExampleR₁-Leu-Asp-Thr-R₂ Prep Calc. Found IC50 1

F 617 617 0.78 2

F 583 583 0.94 3

F 599 599 0.97 4

F 609 609 0.64 5

F 605 605 0.81 6

F 655 655 0.97 7

F 541 541 3.8 8

F 611 611 1.3 9

F 545 545 1.5 10

F 606 617 1.5

EQUIVALENTS

[0210] Those skilled in the art will be able to recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

What is claimed is:
 1. A compound represented by the followingstructural formula: R¹—X—Y′—Z′R² wherein: Y′ is a pentapeptide havingthe sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1), Xaa-Asp-Thr-Ser-Leu(SEQ ID NO: 85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO: 86),Leu-Asp-Xaa-Ser-Leu (SEQ ID NO: 87), Leu-Asp-Thr-Xaa-Leu (SEQ ID NO:88), or Leu-Asp-Thr-Ser-Xaa (SEQ ID NO: 89); Xaa is anaturally-occurring amino acid; X and Z are independently chosen fromthe group consisting of a covalent bond, an amino acid or a peptide,wherein each amino acid in X and Z is independently selected from thegroup of naturally occurring amino acids; R¹ is R³—CO—; R² is —NR⁴R⁵; R³is selected from the group consisting of a lower alkyl group, asubstituted lower alkyl group, an aryl group, a substituted aryl group,a heteroaryl group and a substituted heteroaryl group; R⁴ and R⁵ areeach independently selected from the group consisting of hydrogen, alower alkyl group, a substituted lower alkyl group, an aryl group, asubstituted aryl group, a heteroaryl group and a substituted heteroarylgroup, wherein: 1) R⁴ and R⁵ are not both —H; and 2) taken together, R⁴and R⁵ can form a heterocyclic ring; taken together, X, Y′ and Z form apeptide containing no more than about fifteen amino acids; and whereinoptionally the peptide formed from X, Y′ and Z is cyclized.
 2. Thecompound of claim 1 wherein R³ is selected from the group consisting oftriphenylmethyl, diphenylmethyl, 3,5-diphenylphenyl, 2-furanyl,3-furanyl, 9-xanthenemethyl, 2,2,2-triphenylethyl, 2-anthracene, methyl,cyclopentyl, 2-indolyl, 2-indanyl, 2-benzofuranyl, 3-benzofuranyl,2-benzothienyl, 3-benzothienyl, cyclohexyl, 5-phenylpentyl,4-isobutyl-α-methylphenylmethyl, 4-biphenylmethyl, α-naphthylmethyl,4-heptylphenyl, phenylmethyl, trans 2-phenylethenyl and2,2,3,3-tetramethylcyclopropyl.
 3. The compound of claim 2 wherein R⁴and R⁵ are each independently selected from the group consisting of —H,2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl,3-benzothienyl, -CH₂-2-thienyl, -CH₂-3-thienyl, —CH₂-2-furanyl,—CH2-3-furanyl, 3,4-dimethoxybenzyl, and isopentyl.
 4. The compound ofclaim 3 wherein Y, is Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1).
 5. Thecompound of claim 1 wherein: R³ is selected from the group consisting ofdiphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl,2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and3-benzothienyl; R⁴ is selected from the group consisting of2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl,3-benzothienyl, —CH₂-2-thienyl, —CH₂-3-thienyl, —CH₂-2-furanyl,—CH₂-3-furanyl; and R⁵ is —H.
 6. The compound of claim 5 wherein X and Zare each a covalent bond.
 7. The compound of claim 1 wherein the peptideformed from X, Y, and Z is cyclized.
 8. A compound represented by thefollowing structural formula: R¹—Y′—R² wherein: Y′ is a pentapeptidehaving the sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1),Xaa-Asp-Thr-Ser-Leu (SEQ ID NO:85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO: 86),Leu-Asp-Xaa-Ser-Leu (SEQ ID NO: 87), Leu-Asp-Thr-Xaa-Leu (SEQ ID NO:88), or Leu-Asp-Thr-Ser-Xaa (SEQ ID NO: 89); Xaa is anaturally-occurring amino acid; R¹ is R³—CO—; R² i s —NR⁴R⁵; R³ isselected from the group consisting of a lower alkyl group, a substitutedlower alkyl group, an aryl group, a substituted aryl group, a heteroarylgroup and a substituted heteroaryl group; and R⁴ and R⁵ are eachindependently selected from the group consisting of hydrogen, a loweralkyl group, a substituted lower alkyl group, an aryl group, asubstituted aryl group, a heteroaryl group and a substituted heteroarylgroup, wherein: 1) R⁴ and R⁵ are not both —H; and 2) taken together, R⁴and R⁵ can form a heterocyclic ring; and wherein optionally Y iscyclized.
 9. The compound of claim 8 wherein Y′ has the sequenceLeu-Asp-Thr-Ser-Leu (SEQ ID NO: 1).
 10. The compound of claim 9 whereinR³ is selected from the group consisting of monocyclic and bicyclicnitrogen-containing heteroaromatic groups, vinyl groups substituted withsubstituted and unsubstituted aryl and heteroaryl groups,polycarbocyclic aromatic hydrocarbons and oxygen-containing polycyclicaromatic hydrocarbons.
 11. The compound of claim 9 wherein R³ isselected from the group consisting of a quinolinyl group, anisoquinolinyl group, an indolyl group, a quinoxalinyl group, acinnolinyl group, a pyrazinyl group, a styryl group, a stilbyl group,(3-pyridyl)—CH═CH—, a naphthyl group, an anthracyl group, a xanthanylgroup, a benzopyranone group and a benzofuranyl group.
 12. A compoundrepresented by the following structural formula: R¹—X—Y′—Z—R² wherein:Y′ is a tripeptide [AA]₁-[AA]₂-[AA]₃ having the sequence Leu-Asp-Thr; Xand Z are independently chosen from the group consisting of a covalentbond, an amino acid or a peptide, wherein each amino acid in X and Z isindependently selected from the group of naturally occurring aminoacids; R¹ is R³—CO—; R² is —NR⁴R⁵; R³ is selected from the groupconsisting of a lower alkyl, substituted lower alkyl, aryl, substitutedaryl, heteroaryl and substituted heteroaryl; and R⁴ and R⁵ are eachindependently selected from the group consisting of hydrogen, a loweralkyl group, a substituted lower alkyl group, an aryl group, asubstituted aryl group, a heteroaryl group and a substituted heteroarylgroup, wherein: 1) R⁴ and R⁵ are not both —H; and 2) taken together, R⁴and R⁵ can form a heterocyclic ring; and taken together, X, Y′ and Zform a peptide containing no more than about fifteen amino acids; andwherein optionally the peptide formed from X, Y′ and Z is cyclized. 13.The compound of claim 12 wherein R³ is selected from the groupconsisting of triphenylmethyl, diphenylmethyl, 3,5-diphenylphenyl,2-furanyl, 3-furanyl, 9-xanthenemethyl, 2,2,2-triphenylethyl,2-anthracene, methyl, cyclopentyl, 2-indolyl, 2-indanyl, 2-benzofuranyl,3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, cyclohexyl,5-phenylpentyl, 4-isobutyl-α-methylphenylmethyl, 4-biphenylmethyl,α-naphthylmethyl, 4-hepzylphenyl, phenylmethyl, trans 2-phenylethenyland 2,2,3,3-tetramethylcyclopropyl.
 14. The compound of claim 13 whereinR⁴ and R⁵ are each independently selected from the group consisting of—H, 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl,2-benzothienyl, 3-benzothienyl, -CH₂-2-thienyl, -CH₂-3-thienyl,—CH₂-2-furanyl, —CH₂-3-furanyl, 3,4-dimethoxybenzyl, and isopenzyl. 15.The compound of claim 12 wherein: R³ is selected from the groupconsisting of diphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl,2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and3-benzothienyl; R⁴ is selected from the group consisting of2-hydroxyethyl benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl,3-benzothienyl, —CH₂-2-thienyl, —CH₂-3-thienyl, —CH₂-2-furanyl,—CH₂-3-furanyl; and R⁵ is —H.
 16. The compound of claim 12 wherein thepeptide formed from X, Y′ and Z is cyclized.
 17. A compound representedby the following structural formula: R¹—Y′—R² wherein: Y′ is atripeptide [AA]₁-[AA]₂-[AA]₃ having the sequence Leu-Asp-Thr; R¹ isR³—CO—; R² is —NR⁴R⁵; R³ is selected from the group consisting of alower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryland substituted heteroaryl; and R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, a lower alkyl group, asubstituted lower alkyl group, an aryl group, a substituted aryl group,a heteroaryl group and a substituted heteroaryl group, wherein: 1) R⁴and R⁵ are not both —H; and 2) taken together, R⁴ and R⁵ can form aheterocyclic ring.
 18. The compound of claim 17 wherein R³ is selectedfrom the group consisting of phenyl, substituted phenyl, thienyl,substituted thienyl, indolyl, substituted indolyl, pyrimidyl,substituted pyrimidyl, benzofuranyl, substituted benzofuranyl,quinolinyl, substituted quinolinyl, isoquinolinyl, substitutedisoquinolinyl, benzopyranone groups, substituted benzopyranone groupsand 3-isoquinolinyl-CO—NH—(CH₂)_(x)—, wherein x is an integer from 1-4.19. The compound of claim 18 wherein: R³ is 3-isoquinolinyl or2-benzofuranyl; R⁴ is —H; and R⁵ is benzyl, substituted benzyl,phenethyl, substituted phenethyl, phenpropyl, substituted phenpropyl,heteroaryl-CH₂—, substituted heteroaryl-CH₂—, lower alkyl, substitutedlower alkyl, cycloalkyl and a group represented by one of the followingstructural formulas:


20. The compound of claim 18 wherein: R³ is 3-isoquinolinyl or2-benzofuranyl; and R⁴ and R⁵, taken together, form a heterocyclic ringselected from the group consisting of pyrrolidinyl, substitutedpyrrolidinyl, indoline, isomers of indoline, substituted indoline,substituted isomers of indoline, tetrahydroisoquinoline, substitutedtetrahydroisoquinoline, tetrahydroquinoline, substitutedtetrahydroquinoline, piperidone, substituted piperidone, piperidine,substituted piperidines, tetrahydro-oxazines and substitutedtetrahydro-oxazines.
 21. The compound of claim 17 wherein R¹ isrepresented by the following structural formula:

wherein: A is selected from the group consisting ofi an aryl group, asubstituted aryl group, a heteroaryl group and a substitutedheteroarylgroup; and n and m are each independently zero or one.
 22. Acompound represented by the following structural formula: R¹—X—Y′—Z—R²wherein: Y′ is a dipeptide [AA]₁-[AA]₂ having the sequence Asp-Thr; Xand Z are independently chosen from the group consisting of a covalentbond, an amino acid or a peptide, wherein each amino acid in X and Z isindependently selected from the group of naturally occurring aminoacids; R¹ is R³—CO—; R² is —NR⁴R⁵; R³ is selected from the groupconsisting of a lower alkyl, substituted lower alkyl, aryl, substitutedaryl, heteroaryl and substituted heteroaryl; and R⁴ and R⁵ are eachindependently selected from the group consisting of hydrogen, a loweralkyl group, a substituted lower alkyl group, an aryl group, asubstituted aryl group, a heteroaryl group and a substituted heteroarylgroup, wherein: 1) R⁴ and R⁵ are not both —H; and 2) taken together, R⁴and R⁵ can form a heterocyclic ring; and taken together, X, Y′ and Zform a pentide containing no more than about fifteen amino acids; andwherein optionally the peptide formed from X, Y′ and Z is cyclized withthe proviso that, if the peptide formed from X, Y′ and Z is cyclized,the nitrogen at the N-terminus of Y′ is not bonded to a glycine or asarcosine.
 23. The compound of claim 22 wherein R³ is selected from thegroup consisting of triphenylmethyl, diphenylmethyl, 3,5-diphenylphenyl,2furanyl, 3-furanyl, 9-xanthenemethyl, 2,2,2-triphenylethyl,2-anthracene, methyl, cyclopentyl, 2-indolyl, 2-indanyl, 2-benzofuranyl,3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, cyclohexyl,5-phenylpentyl, 4-isobutyl-α-methylphenylmethyl, 4-biphenylmethyl,a-naphthylmethyl, 4-heptylphenyl, phenylmethyl, trans 2-phenylethenyland 2,2,3,3-tetramethylcyclopropyl.
 24. The compound of claim 23 whereinR⁴ and R⁵ are each independently selected from the group consisting of—H, 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl,2-benzothienyl, 3-benzothienyl, -CH₂-2-thienyl, -CH₂-3-thienyl,—CH₂-2-furanyl, —CH₂-3-furanyl, 3,4-dimethoxybenzyl, and isopentyl. 25.The compound of claim 22 wherein: R³ is selected from the groupconsisting of R³ is selected from the group consisting ofdiphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl,2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and3-benzothienyl; R⁴ is selected from the group consisting of2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl,3-benzothienyl, —CH₂-2-thienyl, —CH₂-3-thienyl, —CH₂-2-furanyl,—CH₂-3-furanyl; and R⁵ is —H.
 26. The compound of claim 25 wherein X andZ are each a covalent bond.
 27. The compound of claim 22 wherein thepeptide formed from X, Y, and Z is cyclized.
 28. A method of treating anindividual suffering from a disease associated with leukocyteinfiltration of tissues expressing the molecule MAdCAM-1, comprisingadministering a therapeutically effective amount of an inhibitorrepresented by the following structural formula: R¹—X—Y—Z—R² wherein: Yis a pentapeptide [AA]₁-[AA]₂-[AA]₃ -[AA]₄-[AA]₅ wherein: [AA]₁ isselected from the group consisting of leucine, cysteine, aspartic acid,glutamic acid, isoleucine, alanine, valine, phenylalanine, glycine,N-methylleucine, serine, threonine, ornithine and lysine; [AA]₂ isselected from the group consisting of aspartic acid, glutamic acid,phenylalanine and tyrosine; [AA]₃ is selected from the group consistingof threonine, serine, valine, proline and 4-hydroxyproline; [AA]₄ isselected from the group consisting of serine, cysteine, aspartic acid,glutamic acid, proline, 4-hydroxyproline, threonine, valine, isoleucine,alanine, glycine, ornithine and lysine; and [AA]₅ is selected from thegroup consisting of leucine, isoleucine, N-methylleucine, threonine,ornithine, serine, valine, alanine, glycine, phenylalanine, cysteine,aspartic acid, glutamic acid and lysine; X and Z are independentlychosen from the group consisting of a covalent bond, an amino acid or apeptide, wherein each amino acid in X and Z is independently selectedfrom the group of naturally occurring amino acids; R¹ is R³—CO—; R² is—NR⁴R⁵; R³ is selected from the group consisting of a lower alkyl,substituted lower alkyl, aryl, substituted aryl, heteroaryl andsubstituted heteroaryl; and R⁴ and R⁵ are each independently selectedfrom the group consisting of hydrogen, a lower alkyl group, asubstituted lower alkyl group, an aryl group, a substituted aryl group,a heteroaryl group and a substituted heteroaryl group, wherein: 1) R⁴and R⁵ are not both —H; and 2) taken together, R⁴ and R⁵ can form aheterocyclic ring; and taken together, X, Y and Z form a peptidecontaining no more than about fifteen amino acids; and wherein:optionally the peptide formed by X, Y and Z is cyclized; and an arginineor an arginine derivative is not bonded to the nitrogen at theN-terminus of Y.
 29. The method of claim 28 wherein Y is a pentapeptidehaving the sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1),Xaa-Asp-Thr-Ser-Leu (SEQ ID NO: 85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO:86), Leu-Asp-Xaa-Ser-Leu (SEQ ID NO: 87), Leu-AsD-Thr-Xaa-Leu (SEQ IDNO: 88), or Leu-Asp-Thr-Ser-Xaa (SEQ ID NO: 89), wherein Xaa is anaturally-occurring amino acid.
 30. The method of claim 29 wherein R³ isselected from the group consisting of triphenylmethyl, diphenylmethyl,3,5-diphenylphenyl, 2-furanyl, 3-furanyl, 9-xanthenemethyl,2,2,2-triphenylethyl, 2-anthracene, methyl, cyclopentyl, 2-indolyl,2-indanyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl,3-benzothienyl, cyclohexyl, 5-phenylpentyl,4-isobutyl-α-methylphenylmethyl, 4-biphenylmethyl, a-naphthylmethyl,4-heptylphenyl, phenylmethyl, trans 2-phenylethenyl and2,2,3,3-tetramethylcyclopropyl.
 31. The method of claim 30 wherein R⁴and R⁵ are each independently selected from the group consisting of —H,2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl,3-benzothienyl, —CH₂-2-thienyl, —CH₂-3-thienyl, —CH₂-2-furanyl,—CH₂-3-furanyl, 3,4-dimethoxybenzyl, and isopentyl.
 32. The method ofclaim 28 wherein: [AA]₁ is selected from the group consisting ofleucine, valine, isoleucine, alanine, glycine, phenylalanine andN-methylleucine; [AA]₂ is selected from the group consisting or asparticacid, glutamic acid, phenylalanine and tyrosine; [AA]₃ is selected fromthe group consisting of threonine, serine, valine, proline and4-hydroxyproline; [AA]₄ is selected from the group consisting of serine,cysteine and threonine; and [AA]₅ is selected from the group consistingof alanine, valine, leucine, isoleucine, alanine, glycine, phenylalanineand N-methylleucine.
 33. The method of claim 32 wherein Y isLeu-Asp-Thr-Ser-Leu (SEQ ID NO: 1).
 34. The method of claim 28 wherein:R³ is selected from the group consisting of diphenylmethyl,triphenylmethyl, trans 2-phenyl-ethylenyl, 2-phenyl-ethynyl,2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and 3-benzothienyl; R⁴ isselected from the group consisting of 2-hydroxyethyl, benzyl,2-benzofuranyl, 3-benzofuranyl, 2-benzoth4enyl, 3-benzothienyl,—CH₂-2-thienyl, —CH₂-3-thienyl, —CH₂-2-furanyl, —CH₂-3-furanyl; and R⁵is —H.
 35. The method of claim 28 wherein the peptide formed from X, Yand Z is cyclized.
 36. A method of treating an individual suffering froma disease associated with leukocyte infiltration of tissues expressingthe molecule MAdCAM-1, comprising administering a therapeuticallyeffective amount of an inhibitor represented by the following structuralformula: R¹—Y′—R² wherein: Y′ is a pentapeptide having the sequenceLeu-Asp-Thr-Ser-Leu (SEQ ID NO: 1), Xaa-Asp-Thr-Ser-Leu (SEQ ID NO: 85),Leu-Xaa-Thr-Ser-Leu (SEQ ID NO: 86), Leu-Asp-Xaa-Ser-Leu (SEQ ID NO:87), Leu-Asp-Thr-Xaa-Leu (SEQ ID NO: 88), or Leu-Asp-Thr-Ser-Xaa (SEQ IDNO: 89); Xaa s a naturally-occurring amino acid; R¹ is R³—CO—; R² is—NR⁴R⁵; R³ is selected from the group consisting of a lower alkyl group,a substituted lower alkyl group, an aryl group, a substituted arylgroup, a heteroaryl group and a substituted heteroaryl group; and R⁴ andR⁵ are each independently selected from the group consisting ofhydrogen, a lower alkyl group, a substituted lower alkyl group, an arylgroup, a substituted aryl group, a heteroaryl group and a substitutedheteroaryl group, wherein: 1) R⁴ and R⁵ are not both —H; and 2) takentogether, R⁴ and R⁵ can form a heterocyclic ring; and wherein optionallyY is cyclized.
 37. The method of claim 36 wherein Y′ has the sequenceLeu-Asp-Thr-Ser-Leu (SEQ ID NO: 1).
 38. The method of claim 37 whereinR³ is selected from the group consisting of a monocyclic and bicyclicnitrogen-containing heteroaromatic groups, vinyl groups substituted withsubstituted and unsubstituted aryl and heteroaryl groups,polycarbocyclic aromatic hydrocarbons and oxygen-containing polycyclicaromatic hydrocarbons.
 39. The method of claim 38 wherein R³ is selectedfrom the group consisting of a quinolinyl group, an isoquinolinyl group,an indolyl group, a quinoxalinyl group, a cinnolinyl group, a pyrazinylgroup, a styryl group, a stilbyl group, (3-pyridyl)—CH═CH—, a naphthylgroup, an anthracyl group, a xanthanyl group, a benzopyranone group anda benzoFuranyl group.
 40. The method of claim 28 wherein the disease isselected from the group consisting of inflammatory bowel disease andinsulin-dependent diabetes mellitus.
 41. A method of treating anindividual suffering from a disease associated with leukocyteinfiltration of tissues expressing the molecule MAdCAM-1, comprisingadministering a therapeutically effective amount of an inhibitorrepresented by the following structural formula: R¹—X—Y′—Z—R² wherein:Y′ is a tripeptide [AA]₁-[AA]₂-[AA]₃ having the sequence Leu-Asp-Thr; Xand Z are independently chosen from the group consisting of a covalentbond, an amino acid or a peptide, wherein each amino acid in X and Z isindependently selected from the group of naturally occurring aminoacids; R¹ is R³—CO—; R is —NRR; R³is selected from the group consistingof a lower alkyl, substituted lower alkyl, aryl, substituted aryl,heteroaryl and substituted heteroaryl; and R⁴ and R⁵ are eachindependently selected from the group consisting of hydrogen, a loweralkyl group, a substituted lower alkyl group, an aryl group, asubstituted aryl group, a heteroaryl group and a substituted heteroarylgroup, wherein: 1) R⁴ and R⁵ are not both —H; and 2) taken together, R⁴and R⁵ can form a heterocyclic ring; and taken together, X, Y′ and Zform a peptide containing no more than about fifteen amino acids; andwherein optionally the peptide formed from X, Y′ and Z is cyclized. 42.The method of claim 41 wherein R³ is selected from the group consistingof triphenylmethyl, diphenylmethyl, 3,5-diphenylphenyl, 2-furanyl,3-furanyl, 9-xanthenemethyl, 2,2,2-triphenylethyl, 2-anthracene, methyl,cyclopentyl, 2-indolyl, 2-indanyl, 2-benzofuranyl, 3-benzofuranyl,2-benzothienyl, 3-benzothienyl, cyclohexyl, 5-phenylpentyl,4-isobutyl-α-methylphenylmethyl, 4-biphenylmethyl, α-naphthylmethyl,4-heptylphenyl, phenylmethyl, trans 2-phenylethenyl and2,2,3,3-tetramethylcyclopropyl.
 43. The method of claim 42 wherein R⁴and R⁵ are each independently selected from the group consisting of —H,2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl,3-benzothienyl, —CH₂-2-thienyl, —CH₂-3-thienyl, —CH₂-2-furanyl,—CH₂-3-furanyl, 3,4-dimethoxybenzyl, and isopentyl.
 44. The method ofclaim 41 wherein: R³ is selected from the group consisting ofdiphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl,2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and3-benzothienyl; R⁴ is selected from the group consisting of2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl,3-benzothienyl, —CH₂-27thienyl, —CH₂-3-thienyl, —CH₂-2-furanyl,—CH₂-3-furanyl; and R⁵ is —H.
 45. The method of claim 42 wherein thepeptide formed from X, Y′ and Z is cyclized.
 46. The method of claim 41wherein the disease is selected from the group consisting ofinflammatory bowel disease and insulin-dependent diabetes mellitus. 47.A method of treating an individual suffering from a disease associatedwith leukocyte infiltration of tissues expressing the molecule MAdCAM-1,comprising administering a therapeutically effective amount of aninhibitor represented by the following structural formula: R¹—Y′—R²wherein: Y′ is a tripeptide [AA]₁-[AA]₂-[AA]₃ having the sequenceLeu-Asp-Thr; R¹ is R³—CO—; R² is —NR⁴R⁵; R³ is selected from the groupconsisting of a lower alkyl, substituted lower alkyl, aryl, substitutedaryl, heteroaryl and substituted heteroaryl; and R⁴ and R⁵ are eachindependently selected from the group consisting of hydrogen, a loweralkyl group, a substituted lower alkyl group, an aryl group, asubstituted aryl group, a heteroaryl group and a substituted heteroarylgroup, wherein: 1) R⁴ and R⁵ are not both —H; and 2) taken together, R⁴and R⁵ can form a heterocyclic ring.
 48. The method of claim 47 whereinR³ is selected from the group consisting of phenyl, substituted phenyl,thienyl, substituted thienyl, indolyl, substituted indolyl, pyrimidyl,substituted pyrimidyl, benzofuranyl, substituted benzofuranyl,quinolinyl, substituted quinolinyl, isoquinolinyl, substitutedisoquinolinyl, benzopyranone groups, substituted benzopyranone groups,and 3-isoquinolinyl—CO—NH—(CH₂)_(x), wherein x is an integer from 1-4.49. The method of claim 48 wherein: R³ is 3-isoquinolinyl or2-benzofuranyl; R⁴ is —H; and R⁵ is benzyl, substituted benzyl,phenethyl, substituted phenethyl, phenpropyl, substituted phenpropyl,heteroaryl-CH₂—, substituted heteroaryl-CH₂—, lower alkyl, substitutedlower alkyl, cycloalkyl, substituted cycloalkyl and a group representedby one of the following structural formulas:


50. The method of claim 48 wherein: R³ is 3-isoquinolinyl or2-benzofuranyl; and R⁴ and R⁵, taken together, form a heterocyclic ringselected from the group consisting of pyrollidine and substitutedpyrrolidinyl, indoline, isomers of indoline, substituted indoline,substituted isomers of indoline, tetrahydroisoquinoline, substitutedtetrahydroisoquincline, tetrahydroquinoline, substitutedtetrahydroquinoline, piperidone, substituted piperidone, piperidine,substituted piperidines, tetrahydro-oxazines and substitutedtetrahydro-oxazines.
 51. The method of claim 47 wherein R¹ isrepresented by the following structural formula:

wherein: A is selected from the group consisting of an aryl group, asubstituted aryl grrouo, a heteroaryl group and a substitutedheteroarylgroup; and n and m are each zero or one.
 52. A method oftreating an individual suffering from a disease associated withleukocyte infiltration of tissues expressing the molecule MAdCAM-1,comprising administering a therapeutically effective amount of aninhibitor represented by the following structural formula: R¹—X—Y′—Z—R²wherein: Y′ is a dipeptide [AA]₁-[AA]₂ having the sequence Asp-Thr; Xand Z are independently chosen from the group consisting of a covalentbond, an amino acid or a peptide, wherein each amino acid in X and Z isindependently selected from the group of naturally occurring aminoacids; R¹ is R³—CO—; R² is —NR⁴R⁵; R³ is selected from the groupconsisting of a lower alkyl, substituted lower alkyl, aryl, substitutedaryl, heteroaryl and substituted heteroaryl; and R⁴ and R⁵ are eachindependently selected from the group consisting of hydrogen, a loweralkyl group, a substituted lower alkyl group, an aryl group, asubstituted aryl group, a heteroaryl group and a substituted heteroarylgroup, wherein: 1) R⁴ and R⁵ are not both —H; and 2) taken together, R⁴and R⁵ can form a heterocyclic ring; and taken together, X, Y′ and Zform a peptide containing no more than about fifteen amino acids; andwherein the peptide formed from X, Y′ and Z is optionally cyclized withthe proviso that, if the peptide formed from X, Y′ and Z is cyclized,the nitrogen at the N-terminus of Y′ is not bonded to a glycine or asarcosine.
 53. The method of claim 52 wherein R³ is selected from thegroup consisting of triphenylmethyl, diphenylmethyl, 3,5-diphenylphenyl,2-furanyl, 3-furanyl, 9-xanthenemethyl, 2,2,2-triphenylethyl,2-anthracene, methyl, cyclopentyl, 2-indolyl, 2-indanyl, 2-benzofuranyl,3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, cyclohexyl,5-phenylpentyl, 4-isobutyl-α-methylphenylmethyl, 4-biphenylmethyl,α-naphthylmethyl, 4-heptylphenyl, phenylmethyl, trans 2-phenylethenyland 2,2,3,3-tetramethylcyclopropyl.
 54. The method of claim 53 whereinR⁴ and R⁵ are each independently selected from the group consisting of—H, 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl,2-benzothienyl, 3-benzothienyl, —CH₂-2-thienyl, —CH₂-3-thienyl,—CH₂-2-furanyl, —CH₂-3-furanyl, 3,4-dimethoxybenzyl, and isopentyl. 55.The method of claim 52 wherein: R³ is selected from the group consistingof diphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl,2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and3-benzothienyl; R⁴ is selected from the group consisting of2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl,3-benzothienyl, —CH₂-2-thienyl, —CH₂-3-thienyl, —CH₂-2-furanyl,—CH₂-3-furanyl; and R⁵ is —H.
 56. The method of claim 55 wherein X and Zare each a covalent bond.
 57. The method of claim 52 wherein the peptideformed from X, Y and Z is cyclized.
 58. The method of claim 52 whereinthe disease is selected from the group consisting of inflammatory boweldisease and insulin-dependent diabetes mellitus.
 59. A method ofinhibiting the binding of a cell expressing a ligand for MAdCAM-1 on thecell surface to MAdCAM-1 or a portion thereof, comprising contacting thecell with an effective amount of an inhibitor represented by thefollowing structural formula: R¹—X—Y—Z—R² wherein: Y is a pentapeptide[AA]₁-[AA]₂-[AA]₃-[AA]₄-[AA]₅ wherein: [AA]₁ is selected from the groupconsisting of leucine, cysteine, aspartic acid, glutamic acid,isoleucine, alanine, valine, glycine, N-methylleucine, serine,threonine, ornithine and lysine; [AA]₂ is selected from the groupconsisting of aspartic acid, glutamic acid, phenylalanine and tyrosine;[AA]₃ is selected from the group consisting of threonine, serine,valine, proline and 4-hydroxyproline; [AA]₄ is selected from the groupconsisting of serine, cysteine, aspartic acid, glutamic acid, proline,4-hydroxyproline, threonine, valine, isoleucine, alanine, glycine,ornithine and lysine; and [AA]₅ is selected from the group consisting ofleucine, isoleucine, N-methylleucine, threonine, ornithine, serine,valine, alanine, glycine, phenylalanine, cysteine, aspartic acid,glutamic acid and lysine; X and Z are independently chosen from thegroup consisting of a covalent bond, an amino acid or a peptide, whereineach amino acid in X and Z is independently selected from the group ofnaturally occurring amino acids; R¹ is R³—CO—; R² is —NR⁴R⁵; R³ isselected from the group consisting of a lower alkyl, substituted loweralkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;and R⁴ and R⁵ are each independently selected from the group consistingof hydrogen, a lower alkyl group, a substituted lower alkyl group, anaryl group, a substituted aryl group, a heteroaryl group and asubstituted heteroaryl group, wherein: 1) R⁴ and R⁵ are not both —H; and2) taken together, R⁴ and R⁵ can form a heterocyclic ring; and takentogether, X, Y and Z form a peptide containing no more than aboutfifteen amino acids; and wherein optionally the peptide formed from X, Yand Z is cyclized.
 60. The method of claim 59 wherein Y is apentapeptide having the sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1),Xaa-Asp-Thr-Ser-Leu (SEQ ID NO: 85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO:86), Leu-Asp-Xaa-Ser-Leu (SEQ ID NO: 87), Leu-Asp-Thr-Xaa-Leu (SEQ IDNO: 88), or Leu-Asp-Thr-Ser-Xaa (SEQ ID NO: 89), wherein Xaa is anaturally-occurring amino acid.
 61. The method of claim 60 wherein theligand is human α4β7 integrin.
 62. The method of claim 61 wherein thecell is a leukocyte.
 63. The method of claim 62 wherein MAdCAM-1 isexpressed on the surface of an endothelial cell.
 64. The method of claim59 wherein the peptide formed from X, Y and Z is cyclized.
 65. A methodof inhibiting the binding of a cell expressing a ligand of MAdCAM-1 toMAdCAM-1 or a portion thereof, comprising contacting the cells with aninhibitory amount of a compound represented by the following structuralformula: R¹—X—Y′—Z—R² wherein: Y is a dipeptide [AA]₁-[AA]₂ having thesequence Asp-Thr or a tripeptide [AA]₁-[AA]₂- [AA]₃ having the sequenceLeu-Asp-Thr; X and Z are independently chosen from the group consistingof a covalent bond, an amino acid or a peptide, wherein each amino acidin X and Z is independently selected from the group of naturallyoccurring amino acids; R¹ is R³—CO—; R² is —NR⁴R⁵; R³ is selected fromthe group consisting of a lower alkyl, substituted lower alkyl, aryl,substituted aryl, heteroaryl and substituted heteroaryl; and R⁴ and R⁵are each independently selected from the group consisting of hydrogen, alower alkyl group, a substituted lower alkyl group, an aryl group, asubstituted aryl group, a heteroaryl group and a substituted heteroarylgroup, wherein: 1) R⁴ and R⁵ are not both —H; and 2) taken together, R⁴and R⁵ can form a heterocyclic ring; and taken together, X, Y′ and Zform a peptide containing no more than about fifteen amino acids; andwherein optionally the peptide formed from X, Y′ and Z is cyclized withthe proviso that, if the peptide formed from X, Y′ and Z is cyclized andif Y′ is Asp-Thr, the nitrogen at the N-terminus of Y′ is not bonded toa glycine or a sarcosine.
 66. The method of claim 65 wherein the ligandis human α4β7 integrin.
 67. The method of claim 66 wherein the cell is aleukocyte.
 68. The method of claim 67 wherein MAdCAM-1 is expressed onthe surface of an endothelial cell.
 69. The method of claim 65 whereinthe peptide formed from X, Y′ and Z is cyclized.
 70. A compoundrepresented by the following structural formula: R¹—Y′—R² wherein: Y′ isa dipeptide [AA]₁-[AA]₂ having the sequence Asp-Thr; R¹ is R³—CO—; R² is—NR⁴R⁵; R³ is selected from the group consisting of a lower alkyl group,a substituted lower alkyl group, an aryl group, a substituted arylgroup, a heteroaryl group and a substituted heteroaryl group; and R⁴ andR⁵ are each independently selected from the group consisting ofhydrogen, a lower alkyl group, a substituted lower alkyl group, an arylgroup, a substituted aryl group, a heteroaryl group and a substitutedheteroaryl group, wherein: 1) R⁴ and R⁵ are not both —H; and 2) takentogether, R⁴ and R⁵ can feorm a heterocyclic ring.